Transmission and scanning electron microscopic study of adepidermal granules of teleosts and amphibia

Summary Granules in the adepidermal space of larvae of Salmo irideus, Hynobius tokyoensis and Rhacophorus buergeri, were observed by transmission and scanning electron microscopy. Adepidermal granules of S. irideus were smooth and spherical structures, those of H. tokyoensis were smooth and spherical, or oval, while in R. buergeri these granules appeared as single or grouped tangled strand-like or starfish-like structures under the scanning electron microscope. These adepidermal granules were spread all over the basal lamina in every animal investigated. The different sizes of adepidermal granules of S. irideus and H. tokyoensis seen under the transmission electron microscope are not the result of differently sectioned faces of granules, but the granules themselves exhibit different sizes. The probable functions of these granules are discussed.     

Ultrastructural cytochemistry of the human adrenal medulla

Summary A cytochemical study of the human adrenal medulla showed that it is made up of two cell types, the adrenaline (A-) and noradrenaline (N-) storing cells. A- and N-storing granules were argentaphobic when ultrathin sections of Araldite-embedded medullae were stained according to the periodic acid-thiocarbohydrazide silver proteinate technique of Thiery. A small amount of glycogen (which disappeared after digestion with alpha amylase) in the form of B-particles, as well as lysosomes were, however, visualized by this technique. The entire core of A granules was markedly positive after ultrathin sections of glutaraldehyde-fixed, glycol methacrylate-(GMA-) embedded medullae were stained with phosphotungstic acid (PTA) at a low pH (0.3). The N granules, in contrast, were mostly unreactive. PTA stained a large part of the Golgi complex of A cells, whereas it generally had no such effect on that of the N cells. In both cell types, the cell coat, lysosomes and multivesicular bodies reacted to PTA. The periodic acid —schiff (PAS) technique showed A but not N granules in semithin sections of GMA- or Araldite-embedded medullae. The PTA and PAS stains were abolished by acetylation, restored by saponification, unchanged by methylation and greatly diminished by sulfation or by digestion with beta glucuronidase after oxidation by perchloric acid. These results indicate that in man the A granules and the Golgi complex of A cells, unlike the same structures in N cells, are rich in glycoproteins.     

Biochemical study of the adepidermal granules of Urodela and Anura—Especially on lipids

Summary The lipids of the adepidermal granules of Hynobius tokyoensis and Rana japonica were analysed qualitatively. To extract the lipids from the adepidermal granules, the epidermis of the larvae of both animals were removed from the body. Then the granules were extracted from the surface of the basal lamina by means of wiping with the cotton balls containing the solvent. It was ascertained with the electron microscope whether the adepidermal granules were extracted by the cotton balls or not.By the analysis which was made with thin layer chromatography, the same kind of phospholipids or neutral lipids were detected from the extracts of both R. Japonica and H. tokyoensis. Moreover, four spots which seemed to contain some sialic acids were detected from the both extracts.     

ULTRASTRUCTURAL CYTOCHEMISTRY : Enzyme and Acid Hydrolysis of Nucleic Acids and Protein

Selective extraction of specific cell components by enzyme or acid hydrolysis is possible from ultrathin sections for electron microscopy and parallel 2 µ sections for light microscopy of tissues fixed in formalin and embedded in a water-soluble polyepoxide, product X133/2097. Normal rat tissues fixed 15 minutes in formalin at 3°C are more rapidly digested by proteinases than those fixed for the same length of time at 20°C. Trypsin selectively attacks the nuclear chromatin and the ribonucleoprotein particles of the ergastroplasm, whereas mitochondria and zymogen granules resist tryptic digestion. Pepsin rapidly attacks the mitochondria and zymogen granules. The ergastoplasm and nucleus at first resist peptic digestion, but in time the entire cytoplasm and interchromatinic portion of the nucleus are attacked. Ribonuclease abolishes cytoplasmic basophilia in 2 µ sections, but parallel ultra-thin sections, stained with uranyl acetate and examined in the electron microscope, show no change in the ribonucleoprotein particles of the ergastoplasm. Desoxyribonuclease alone had no effect, but after pretreatment of the sections with pepsin or hydrochloric acid, desoxyribonuclease specifically attacked the nuclear chromatin. Nucleic acid-containing structures in the sections are gradually disintegrated by perchloric acid or hydrochloric acid.     

Fine structure and cytochemistry of specific granules in the lamprey atrium

Summary Fine structural and cytochemical studies were performed to examine the nature of three types of specific granules found in the atrium of lamprey; specific granules of the atrial muscle cell (ASG), interstitial cell granules (ICG) and endocardial endothelial granules (ESG).Ultrastructurally, ASG and ICG appeared quite similar in size, shape and electron opacity, while ESG were much larger and less dense in opacity than the other two.None of the granules showed positive DAB reaction or acid phosphatase reaction. Only ICG revealed positive chromaffin reaction, which agreed with formaldehyde induced green fluorescence along the atrial lumen. Phosphotungstic acid at low pH stained ICG and ASG strongly positive, and ESG weakly positive. Pronase treatment in Epon sections for 24h digested ASG alone, whereas in glycol-methacrylate embedded sections, ESG were digested first, ASG were digested thoroughly after 30 min, but ICG were not digested completely after 90 min.From these results it can be concluded that the three types of specific granules have different constituents. ESG consist of protein with some polysaccharides; ASG are composed of protein carbohydrate complexes and lack catecholamines; ICG contain catecholamine as well as protein carbohydrate complexes.This work was supported by a grant from the Ministry of Education, JapanThe authors would like to express their gratitude to K. Wasano, M.D. for his technical assistance in fluorescence microscopy     

Myelin protein changes with digestion of whole sciatic nerve in trypsin.

Whole mouse sciatic nerves were split and incubated in phosphate buffered saline (PBS) and in PBS containing various amounts of trypsin. After 24 hours of exposure to PBS alone there were no changes in the gel electrophoresis pattern of myelin proteins. During the same period of time, trypsin digested major amounts ofboth the main myelin protein (P_O) and the two basic proteins of myelin (P₁, P₂). The basic proteins were undetectable after 24 hours of 1% trypsin digestion while the main myelin protein was not completely digested. The amount of digestion of the myelin proteins was related to the concentration of trypsin and the time of digestion. Myelin proteins were demonstrated by staining with Coomassie blue, periodic acid Schiff (PAS) and by special indirect lighting techniques.     

Evaluation of the glycogenolytic effect of alpha-amylase using radioautography and electron microscopy.

The effectiveness of crystalline alpha-amylase and saliva in hydrolyzing newly formed glycogen in liver and muscle was examined. Glycogen synthesis was induced by the administration of H3-glucose to fasting rats or by the incubation of tissue slices in a medium containing H3-glucose. Paraffin sections of Rossman-fixed tissues or small pieces of liver fixed in glutaraldehyde and subsequently postosmicated and embedded in Epon were then enzymatically digested. Grain counts were made in radioautographs of treated and untreated materials, and the amount of radioactivity removed by the digestion was used to assess the efficiency of the enzymes in hydrolyzing glycogen. Crystalline alpha-amylase hydrolyzed almost completely newly formed glycogen in liver and muscle. Saliva removed the glycogen that was synthesized in vivo, but it was less effective in hydrolyzing glycogen synthesized in vitro. Electron micrographs of digested liver cells confirmed the radioautographic findings on the effectiveness of the enzyme preparations.     

Glycoproteins in membranes of secretory granules of the anterior pituitary gland.

Rat anterior pituitary glands were examined by electron microscopy after staining with five different histochemical stains. Histochemical reactions were observed in the cell coat, cell membrane and the membrane surrounding the secretory granules in all anterior pituitary cells following staining with phosphotungstic acid (PTA), chromic acid and PTA, the periodic acid-thiosemicarbazide-silver protein method (PA-TSC-SP) of Thiéry, ruthenium red and concanavalin A. The staining was abolished when the sections were preincubated with pronase, neuraminidase or trypsin and subsequently exposed to PTA, chromic acid and PTA or PA-TSC-SP. The possible functional role of the glycoproteins present in the membrane surrounding the secretory granules is considered.     

The role of two isoenzymes of alpha-amylase of Araucaria araucana (Araucariaceae) on the digestion of starch granules during germination

Starch is the principal reserve of Araucaria araucana seeds, and it is hydrolysed during germination mainly by alpha-amylase. There are several alpha-amylase isoenzymes whose patterns change in the embryo and in the megagametophyte from the one observed in quiescent seeds (T(0)) to a different one observed 90 h after imbibition (T(90)). The objective of this research was to study the roles of two purified alpha-amylase isoenzymes by in vitro digestion of starch granules extracted from the tissues at two times of imbibition: one is abundant in quiescent seeds and the other is abundant after 90 h of imbibition. The isoenzymes digested the starch granules of their own stage of germination better, since the isoenzyme T(0) digested starch granules mainly from quiescent seeds, while the isoenzyme T(90) digested starch mainly at 90 h of imbibition. The sizes of the starch granule and the tissue from which these granules originated make a difference to digestion by the isoenzymes. Embryonic isoenzyme T(0) digested large embryonic starch granules better than small and medium-sized granules, and better than those isolated from megagametophytes. Similarly isoenzyme T(90) digested small embryonic starch granules better than medium-sized and large granules, and better than those isolated from megagametophytes. However, a mixture of partially purified megagametophytic isoenzymes T(0) and T(90) digested the megagametophytic granules better than those isolated from embryos. Studies of in vitro sequential digestion of starch granules with these isoenzymes corroborated their specificity. The isoenzyme T(90) digested starch granules previously digested by the isoenzyme T(0). This suggests that in vivo these two isoenzymes may act sequentially in starch granule digestion.     

Fine Structure of Listeria monocytogenes in Relation to Protoplast Formation

Among the eight strains of Listeria monocytogenes tested for lysozyme sensitivity, two were resistant to lysozyme but became sensitive after lipase pretreatment. Among the other six, one was very sensitive to lipase and another one was extremely susceptible to lysozyme. Stable protoplasts were formed from the lysozyme-resistant strain (42) by lipase and lysozyme treatment, which completely digested the cell wall. The cell wall (uranyl acetate-lead stained) was of a thick triple-layered profile, with the intermediate layer of low density. Lipase treatment for a short time (60 min) did not cause any alteration in structure, but prolonged treatment (180 min) caused extensive digestion of the plasma membrane and the cell wall, liberating cytoplasmic material. When the cells were treated with either lipase or lysozyme, a small number of protoplasts were extruded through the partly digested or weakened transverse cell wall, leaving an almost intact cell wall ghost. There were small vesicular structures in the interspace between cell wall and plasma membrane. Mesosomes of varied organization were prominent in electron micrographs, both in sections and in negatively stained preparations. These were largely everted during protoplasting in the form of tubules and as small peripheral buds; a few small vesicles also remained as intrusive structures, some of which were very unusual because they appeared to be enclosed by the inner layer of plasma membrane alone. Lysis of the protoplasts by dilution of the sucrose, while maintaining a constant ionic environment, liberated many small vesicular structures and fibrillar nuclear material.     

The uptake and utilization of Entodinium caudatum, bacteria, free amino acids and glucose by the rumen ciliate Entodinium bursa

C oleman , G.S. & H all , F.J. 1984. The uptake and utilization of Entodinium caudatum , bacteria, free amino acids and glucose by the rumen ciliate Entodinium bursa. Journal of Applied Bacteriology 56 , 283–294.
Washed suspensions of Entodinium bursa were incubated anaerobically with Entodinium caudatum , ten species of bacteria and a yeast. The rate of uptake and digestion of these micro-organisms was investigated. Protozoa grown in vivo did not engulf Proteus mirabilis or Klebsiella aerogenes but rapidly took up Bacillus mega-terium, Selenomonas ruminantium, Torulopsis glabrata and Streptococcus bouis , although only the last was digested with release of soluble material into the medium. Protozoa grown in vitro engulfed each of the bacteria tested, taking up Megasphaera elsdenii and i>Proteus mirabilis most rapidly. Individual bacterial species and mixed rumen bacteria were engulfed more rapidly (up to 20 times) by protozoa grown in vivo than those grown in vitro , although the latter digested over 80% of the B. megaterium, Escherichia coli and P. mirabilis taken up. Labelled Ent. caudatum was extensively digested after engulfment by Ent. bursa . Some of the digestion products were released into the medium but individual amino acids were transferred as such from Ent. caudatum protein to Ent. bursa protein. Engulfed bacteria and polysaccharide granules were transferred intact from one protozoon to the other. Free amino acids were also taken up intact from the medium into protozoal protein but there was little biosynthesis of amino acids from glucose. When available for engulfment Ent. caudatum was quantitatively a much more valuable source of amino acids for protein synthesis by Ent. bursa than free amino acids or bacteria.     

Secretory granules of B-cells in the synovial membrane

Summary Ultrastructural and cytochemical studies have been made on secretory granules of B-cells (fibroblast-like cells) in the knee-joint synovium. The secretory granules were membrane-bounded spherical or slightly elongated bodies, 150 to 350 nm (average 230 nm) in diameter and had a homogenous matrix with several cores. These granules were found in B-cells of all animal species examined; they were numerous in mice and rats, and few in guinea pigs, rabbits and man. Ultrastructural and cytochemical examinations revealed that the Golgi apparatus was involved in the formation of the secretory granules. Unlike lysosomes, they showed no acid phosphatase activity. The granule matrix was positively stained by Thiéiy's periodic acid-thiocarbohydrazidesilver proteinate technique, and the cores were digested by protease. These findings suggest that the granule matrix contains mucopolysaccharide(s) and/or glycoprotein(s) and the core material is largely proteinaceous in nature.     

Phenylalanine metabolism in isolated rat liver cells. Effects of glucagon and diabetes.

Tryptic digestion of histone H1 from the sperm of the sea urchin Sphaerechinus granularis leaves a limiting peptide of approx. 80 residues that is of similar size to the limit peptide from calf thymus H1 or chicken erythrocyte H5. The S. granularis limit peptide folds to form tertiary structure similar to that of the intact parent histone H1 (shown by n.m.r. spectra), but the helical content is decreased by the digestion from 64 residues to 28. In contrast, intact calf thymus H1 and chicken erythrocyte H5 histones have only about 28 helical residues, which are preserved in their limit peptides. The extra helix in S. granularis is shown to be rapidly digested away by trypsin, and its location in histone H1 is discussed. A possible relationship of this structural feature to the length of linker DNA is proposed.     

EFFECT OF LIPASE-DIGESTION ON PHOTOCHEMICAL ACTIVITIES OF ISOLATED CHLOROPLASTS

In order to elucidate the role of lipids in photosynthesis,chloroplasts were digested with lipase, and the effect of lipase-digestionon some photochemical activities was studied. The HILL reactionwas sensitive to the digestion, but chloroplasts having intactmembrane were somewhat resistant to the action of lipase. Theinactivation by lipase digestion seems to be due to the destructionof a component necessary for the Hill reaction to proceed. Thechloroplasts treated with lipase showed the following activities. (1) Active photooxidation of reduced cytochrome c and menadione. (2) Photooxidation of ascorbate, which was enhanced in the presenceof DPIP, and retarded in the absence of the dye. (3) NADP-photoreduction in the presence of the DPIP-ascorbatecouple, as the electron donor. These facts suggested that the site attacked with lipase wasresponsible for the photochemical oxygen evolution. The decrease in the fluorescence intensity of chlorophyll awas also observed during the digestion. ¹Present address : Biological Laboratory, General EducationDeparment, Kyushu University, Otsubo-machi, Fukuoka.     

Sex and strain differences in the brush border and PAS-positive granules and giant bodies of the mouse renal S3 segment cells.

We recently demonstrated sexual dimorphism in the S3 segment of the ICR mouse kidney, as differences in periodic acid Schiff (PAS) staining on the brush border and the number and size of PAS-positive granules. However, whether these sex dependent features in the S3 segment of the mouse kidney occur only in the ICR strain or are a general feature also observed in other strains is unclear. In the present study, we examined the renal S3 segment of the ICR, BALB/c, C57BL/6, C3H/He and DBA/2 mice strains, which are commonly used in laboratory experiments. PAS staining of the brush border in females of all strains was more intense than that of males, and PAS-positive granules were detected in all females. In male groups, PAS-positive granules were detected in the DBA/2 strain only, but their number was very few. In addition, PAS-positive giant bodies, larger than the nuclear size, were detected in females except those of the C57BL/6 strain. Histometrical investigation demonstrated apparent strain differences in a number of PAS-positive granules and PAS-positive giant bodies. The ultrastructural and cytochemical investigations suggest that the PAS-positive granules and PAS-positive giant bodies were multilamellar lysosomes. We propose that the present findings are significant for comparative morphology in laboratory animal science.     

Endoribonuclease from bovine adrenal cortex cytosol.

An endoribonuclease which digests a variety of synthetic homoribopolymers and poly(A)-rich mRNA has been identified and purified greater than 500-fold with respect to specific activity from bovine adrenal cortex cytosol. Enzymatic digestion of synthetic poly(riboadenylic acid) was stimulated by Mn-2+ and Mg-2+ and the enzyme exhibited broad pH and salt optima. Poly(cytidylic acid) and poly(uridylic acid), but not poly(guanylic acid), served as substrates for the enzyme preparation; double-stranded RNA, DNA, and DNA-RNA hybrids were not digested by the enzyme. Digestion generated oligonucleotides with 3-hydroxyl and 5'-monophosphoester termini. On isoelectric focusing, the enzymatic activity banded at pH 8.3 plus or minus 0.2. An initial preferential cleavage of the poly(A) tract of poly(A)-rich RNA is suggested by the rapid appearance of a 4-6S digestion product highly enriched for adenylic acid; however, progressive digestion of the RNA occurs with additional incubation.     

Histochemistry of glycogen in the inner ear

Synopsis The effect of fixation and processing upon the morphological appearance of glycogen within the outer hair cells of the guinea-pig was investigated using two methods. In each method, tissue was fixed for 12 h in cold phosphate-buffered 4% paraformaldehyde and eventually dehydrated in ethanol, embedded in Epon 812, and cut into 4 m sections. In procedure A, after complete processing, the sections were tained using the periodic acid-Schiff reaction (PAS) or the periodic acid-thiocarbo-hydrazide-osmium tetroxide (PATCO) reaction which resulted in the appearance of listinct, coarse granules in the cytoplasm of the outer hair cells. Diastase digestion on one of the two matched sections after Epon removal and prior to staining, confirmed the granules to be glycogen. In procedure B, after primary fixation, the tissue was post-fixed in 1% osmium tetroxide and then processed exactly as in procedure A. Here, unless the Epon and osmium was remoyed, there was no staining of the outer hair cell cytoplasm. However, after Epon removal there was diffuse, grainy appearance of the outer hair cell cytoplasm which we considered to be due to glycogen although diastase confirmation was not possible. We have concluded that osmium tetroxide (1) inhibits PAS or PATCO staining, (2) prevents diastase digestion, and (3) prevents the appearance by light microscopy of distinct granules of glycogen.     

Post-embedding staining of rat gastric mucous cells with lectins

Summary The mucous cells of the rat stomach were stained with lectins by two post-embedding staining methods for electron microscopy. The mucous granules of surface mucous cells and foveolar mucous cells were stained weakly by Ricinus communis agglutinin-ferritin and wheat germ agglutinin-ferritin. The mucous granules of mucous neck cells were stained by concanavalin A-ferritin, Ricinus communis agglutinin-ferritin and wheat germ agglutinin-ferritin. The mucous granules of pyloric gland cells showed an affinity for wheat germ agglutinin-ferritin and concanavalin A-ferritin, while Ricinuscommunis agglutinin-ferritin only slightly stained the granules. The granules of mucous neck cells and pyloric gland cells were also stained by the concanavalin A-horseradish peroxidase-colloidal gold method, but the granules of surface and foveolar mucous cells were not stained by this method. Periodic acid oxidation of the sections before the standard concanavalin A-ferritin procedure enhanced the staining of the granules of mucous neck cells and pyloric gland cells slightly. Reduction of the sections after the periodic acid oxidation weakened the staining. Similar results were obtained using the concanavalin A-horseradish peroxidase-colloidal gold method. Though the staining with Ricinus communis agglutinin-ferritin was inhibited by periodic acid oxidation of the sections before staining, the staining with wheat germ agglutinin-ferritin was not inhibited by the oxidation. It is suggested that the paradoxical staining is closely related to the position of the concanavalin A-binding sugar residues in the carbohydrate chains.This work was supported in part by a grant-in-aid (No. 457008) from the Ministry of Education, Science and Culture, Japan and a grant-in-aid for cancer research (55-21) from the Ministry of Health and Welfare, Japan     

THE PROTEIN COMPOSITION OF NUCLEAR CRYSTALS IN LEAF CELLS

The chemical composition of perinuclear crystals of Lychnis chalcedonica and intranuclear crystals of Dianthus barbatus in leaf tissue embedded in glycol methacrylate was tested by differential enzyme digestion of ultra-thin sections. The Lychnis crystals were digested readily by pepsin but with great difficulty and never to completion by trypsin. The Dianthus crystals were rapidly and completely digested by pronase; inconsistently and with difficulty by pepsin; and were unaffected by trypsin. Pre-incubation with water increased the effectiveness of pepsin digestion. Neither RNase nor DNase had any effect, either alone, or when preceded or followed by the proteinases. It was concluded that the Lychnis crystals are composed of protein that is probably high in aromatic amino acid content, and possibly low in the basic amino acids arginine and lysine. The Dianthus crystals also seem to be protein, but because of their amino acid composition or conformation, and possibly because of complex reactions with the fixative, these crystals are not readily hydrolyzed by pepsin and trypsin.     

Digestion of Barley, Maize, and Wheat by Selected Species of Ruminal Bacteria

Differences in the digestion of barley, maize, and wheat by three major ruminal starch-digesting bacterial species, Streptococcus bovis 26, Ruminobacter amylophilus 50, and Butyrivibrio fibrisolvens A38, were characterized. The rate of starch digestion in all cereal species was greater for S. bovis 26 than for R. amylophilus 50 or B. fibrisolvens A38. Starch digestion by S. bovis 26 was greater in wheat than in barley or maize, whereas starch digestion by R. amylophilus 50 was greater in barley than in maize or wheat. B. fibrisolvens A38 digested the starch in barley and maize to a similar extent but was virtually unable to digest the starch in wheat. The higher ammonia concentration in cultures of B. fibrisolvens A38 when grown on wheat than when grown on barley or maize suggests that B. fibrisolvens A38 utilized wheat protein rather than starch. Scanning electron microscopy revealed that B. fibrisolvens A38 initially colonized cell wall material, while S. bovis 26 randomly colonized the endosperm and R. amylophilus 50 preferentially colonized starch granules. There was subsequent colonization but only superficial digestion of wheat starch granules by B. fibrisolvens A38. Variation in the association between starch and protein within the endosperm of cereal grains contributes to the differential effectiveness with which amylolytic species can utilize cereal starch.