Protargol Research Articles

Electron microscopic cytochemical study of cell-wall polysaccharides in Bacillus subtilis and two strains of Bacillus megaterium

Thin sections of Bacillus subtilis and B. megaterium cells were treated by two cytochemical techniques revealing polysaccharides: the phosphotungstic-acid (PTA) and the silver-proteinate (SP) stainings. These procedures were applied to exponentially growing cells as well as cells treated with chloramphenicol (CMP) or cells regrown after antibiotic elimination. It appeared that both stains mainly deposited on the cell wall, but did not give the same staining patterns. In vegetative and CMP-treated cells, PTA gave a homogenous dark deposit, whereas with SP a deposit was only observed on the outer and inner wall faces. During cell-wall thinning, PTA-stained walls remained rather homogeneous, whereas the silver deposit spread throughout the cell-wall thickness. The meaning of these differences in relation to cell-wall polymer organization is discussed.

Ultrastructural Localization of Polysaccharides in Apple Leaf Cuticles1,2

Abstract The ultrastructural localization of polysaccarides in Malus domestica Borkh. leaf cuticles was investigated by electron microscopy staining. The cuticle/cell wall interface was not stained by ruthenium red or hydroxylamine-ferric chloride, indicating the lack of a distinct pectin layer. The inner region of the cuticle was intensely and uniformly stained by phosphotungstic acid (PTA) and was lightly stained by silver proteinate, indicating the presence of polysaccharides, which in part may be pectin as indicated by staining with ruthenium red. Fibrils in the inner region of the cuticle were not stained by ruthenium red, hydroxylamine-ferric chloride or silver proteinate, but appeared lightly stained by PTA; this suggests by deduction that the fibrils may consist of cutinized cellulose. The outer region of the cuticle was not stained by any of the staining procedures, indicating the lack of polysaccharides.

The efforts of drugs on ciliary motility II. Antimicrobial agents

The effects of antimicrobial agents at different pHs on the ciliary beat frequency of chicken embryo tracheas have been investigated. Benzylpenicillin sodium (10,000 U/ml), ampicillin sodium (1%), neomycin sulphate (0.35%), polymyxin (0.1%), sulphanilamide sodium (0.4%) and mild silver protein (0.5%) do not decrease the ciliary beat frequency by more than 50% in 1 h. Chloramphenicol (0.4%), bacitracin (10,000 U/ml) and sulphacetamide sodium (10%) arrest ciliary movement within 1 h, and only the effects of bacitracin were not reversible. Lowering the pH of the antibiotic-containing solutions aggravated their ciliotoxicity in proportion to the increase in effect of lowering the pH in agent-free Locke Ringer solution.

Substructure of granules from serous cells of porcine tracheal submucosal glands.

The ultrastructure of serous cells from porcine tracheal submucosal glands was studied by conventional transmission electron microscopy (TEM), and by cytochemical methods to stain for complex carbohydrates. In tissue fixed and processed for TEM, and stained with uranyl acetate and lead citrate, the condensing granules of serous cells occasionally possessed a hexagonal and sometimes a lamellar substructure. Tissue fixed in paraformaldehyde-glutaraldehyde and stained with periodic acid-thiocarbohydrazide-silver proteinate (PTS) or with phosphotungstic acid (PTA) showed secretory granules stained for complex carbohydrates and revealed a substructure similar to that noted in the condensing granules. The dark staining substructure revealed by either the PTS or the PTA technique appeared to correspond to electron-lucent areas observed in the condensing granules by conventional TEM. The PTS staining probably demonstrated the presence of neutral glycoprotein, since the serous-cell granules did not react with a dialyzed iron stain for acidic glycoproteins. Treatment of periodic acid oxidized thin sections with pronase or pepsin prior to thiocarbohydrazide and silver proteinate treatment decreased the intensity of the PTS staining, but did not digest away any components of the granules. The substructure revealed by the carbohydrate stains may be a reflection of the mechanism of packaging or the macromolecular structure of the glycoproteins in the serous-cell granules.

Rat liver metabollothionein interactions of silver, zinc, and cadmium

Weanling rats were fed diets either alone or with combinations of silver, elevated zinc, or elevated cadmium for 7 weeks. The rats were then killed, and the silver, zinc, and cadmium proteins isolated from the livers by gel filtration and ion exchange chromatography. When silver was fed alone in diet, low levels of this metal were eluted as two peaks from the ion exchange column. In contrast, when silver was fed with cadmium or elevated zinc, three metal-containing peaks were eluted from this ion exchange column. Amino acid analysis revealed that the major proteins binding these metals are metallothioneins, as judged by high cysteine content.

Simplified silver protein detection and image enhancement methods in polyacrylamide gels

AbstractThe use of silver for detection of protein in polyacrylamide gels is becoming widespread. An effort has been made to develop a silver stain which minimizes the time to perform the stain, the amount of silver used, and the complexity of the procedures, and which maximizes the sensitivity. This stain is almost 200‐fold more sensitive than the Coomassie Brilliant Blue R‐250 stain. This silver stain procedure has been shown, with eight purified proteins, to generally be linear over a 40‐fold range in concentration, between 0.005 ng/mm2 to 2.0 ng/mm2. For greater sensitivity, a recycling procedure has been developed. This procedure is capable of amplifying trace proteins which could not be visualized with previous silver stain techniques. Analysis of the kinetics of image development revealed that the silver ion was the limiting factor in image formation. By recycling the gel through silver nitrate solutions, silver ions are replenished in the gel, permitting amplification of further details when the gel is treated with a developing solution. A number of problems inherent in silver stain detection of proteins in polyacrylamide gels are discussed with suggested remedies.

Cell surface carbohydrates in Tritrichomonas foetus.

The cell surface of Tritrichomonas foetus was characterized by using 18 highly purified lectins with specificities for N-acetyl glucosamine, N-acetyl galactosamine, galactose, mannose, and sialic acid. The specificity of the lectin-induced cell agglutination was verified by inhibition of the agglutination with the specific sugars. By using cytochemical techniques associated with electron microscopy, carbohydrates were detected on the cell surface of T. foetus. The following techniques were used: periodic acid--thiosemicarbazide--silver proteinate, concanavalin A--horseradish peroxidase, and ruthenium red. Anionic sites were detected on the cell surface of the protozoan at pH's 1.8 and 7.2 with the use of colloidal iron hydroxide and cationized ferritin particles, respectively. The binding of colloidal iron particles, as well as the agglutination induced by the lectin from Limulus polyphemus, indicated the presence of sialic acid on the cell surface of T. foetus.

The appearance of carbohydrate-rich material in the developing Golgi apparatus of amoebae.

The silver proteinate reaction was used to stain carbohydrate-rich substances in normal Amoeba proteus and in the developing Golgi apparatus of renucleated amoebae. Normal cells contained stained material, which probably is glycoprotein, in the cell surface, cisternae at the concave pole of the Golgi apparatus, and cytoplasmic vesicles and vacuoles. Previous radioautographic studies had shown tht glycosylation occurs in the Golgi apparatus, and that material in the Golgi apparatus is precursor to the cell surface. Amoebae were enucleated for 5 d, which results in a decline of the Golgi apparatus, the disappearance of the glycoprotein-containing cisternae preceding that of the rest of the organelle. A new nucleus was then transplanted into the enucleate amoebae, bringing about the regeneration of the Golgi apparatus. small curved cisternae that appeared 30 min after renucleation lacked staining with silver proteinate. By 1 h after renucleation, however, the content of cisternae toward the concave poles of Golgi bodies stained with silver proteinate. The Golgi apparatus in cells fixed 6 h and 1 d after operation resembled that of normal amoebae in both morphology and staining pattern. The results suggest that the developing Golgi apparatus acquired the capacity to participate in assembly of cell-surface material within 1 h after renucleation. This occurred before development of the normal enzymic activity of the Golgi apparatus was completed.

The effect of chloroquine on the intralysosomal degradation of cell-coat glycoproteins in the absorptive cells of cultured human small-intestinal tissue as shown by silver proteinate staining.

The effect of chloroquine on the intralysosomal degradation of cell-coat glycoproteins in cultured intestinal absorptive cell was investigated by silver proteinate staining. The results of this staining method, which is specific for carbohydrate containing macromolecules such as glycoproteins and mucopolysaccharides, showed that in the presence of the drug considerable amounts of silver proteinate-positive material accumulated in one type of lysosome-like body: the dense bodies. The staining pattern of other cell organelles was not affected by chloroquine. The presence of the drug in the culture medium also resulted in the occurrence of numerous small vesicular structures in the matrix of the dense bodies. These showed a similar size and structure to those present in the other type of lysosome-like body: the multivesicular bodies. This observation, together with earlier autoradiographical data, suggests that cell-coat material is transferred from multivesicular to dense bodies by fusion between these organelles. This study thus provides further evidence for a regulatory mechanism of cell-coat glycoprotein transport by the lysosome-like bodies in human intestinal absorptive cells.

MUCOSUBSTANCES OF THE RAT GASTRIC MUCOSA STUDIED BY MEANS OF SEVERAL HISTOCHEMICAL METHODS

Distribution of mucosubstances in the rat gastric mucosa was studied by radioautography using 35S-sodium sulfate, several light microscopic histochemical procedures, the periodic acid thiocarbohydrazide silver proteinate (PA-TCH-SP) method and postembedding staining with Iectin (wheat germ agglutinin) ferritin conjugates. Histochemical staining revealed that the superficial covering epithelium contained a large amount of neutral mucosubstances, whereas the foveolar and isthmus mucous cells produced both sialomucins and sulfomucins. Active incorporation of 35S was observed in the fundic foveolus and isthmus as well as the pyloric gland. The mucous neck cells contained sialomucin. The PA-TCH-SP method stained the covering epithelium, the epithelium of the foveolar and isthmus mucous cells and the mucous neck cells. All cell organellae which took part in the formation of granule substances in the gastric mucosa were stained by this method. The cytoplasm of the mucous neck cells was extensively occupied with round PA-TCH-SP positive granules. As the epithelium of the pits matured, the granules increased in size. The apical portion of the covering epithelium was exclusively occupied with large round to oval granules. Many of them showed a bipartite structure after PA-TCH-SP staining, reaction products were observed only in the peripheral region of these granules. Conversely, the development of other cell organellae became less prominent as these epithelia matured. Postembedding staining with lectin (wheat germ agglutinin)-ferritin conjugates stained secretory granules of all these mucous cells with different staining patterns. In the covering epithelium, the conjugate stained only the peripheral region of the granules.

The collagen gland of Mytilus galloprovincialis: An ultrastructural and cytochemical study on secretory granules

The collagen gland of the byssal apparatus of Mytilus sp. is a quite exceptional case of an exocrine collagen-secreting gland. The ultrastructural study demonstrates that collagen gland cells produce two kinds of secretory granules containing the same structural elements as the byssus threads, i.e., a microfilamentous matrix, 7- to 9-nm-thick filaments, and filamentous banded elements. Cytochemical methods at the ultrastructural level (phosphotungstic acid at low pH, periodic acid—silver methenamine, thiosemicarbazide—silver proteinate, silver methenamine for sulfhydryl group demonstration) have shown that secretory granules are mainly proteinaceous, are poor in carbohydrates, and contain SH or S—S groups. Moreover the results of enzyme digestion (collagenase, pepsin, trypsin, α-chymotrypsin) indicate that collagen is mainly localized in the microfilamentous matrix and that it is only a component of a proteinaceous complex. Byssus collagen molecules are organized in the final secretory product intracellularly, unlike what is known to occur in other collagen-secreting cells.

LITERATURE ABSTRACTS

GENERAL PRINCIPLES: Effects of Disulfide Bond on the Gel Formation of Myosin B and/or Soy Protein. CIF. Seiichi Haga and Iomio Ohashi.GENERAL PRINCIPLES: Effects of Ca2+ and Mg2+ on Gel Formation of a Mixture of Myosin B with Soy Protein CIF. Seiichi Haga and Tomio Ohashi.GENERAL PRINCIPLES: The Effect of Heat Processing on the Structure and Rheological Properties of Carrageenan Gels. P. A. Ainsworth and J. M. V. Blanshard.GENERAL PRINCIPLES: Observation of Internal Structure of Casein Submicelles by Means of Ion BeamINSTRUMENTATION AND METHODOLOGY: A Sca!?Ziilg Electron Microscopy Stady of Japanese Noodles. J. E. Dexter and B. L. Dronzer.INSTRUMENTATION AND METHODOLOGY: Apparatus for Monitoring Cake Structure Development During Baking. P. W. Voisey.INSTRUMENTATION AND METHODOLOGY: An Evaluation of the Rapid Amylograph Method. B. A. Marchylo and F. G. Kosmdak,INSTRUMENTATION AND METHODOLOGY: Silver Proteinate Staining of Neutral Polysaccharides in Apple Cell Walls: Implications Relative to Fruit Firmness. W. P. Mohr.INSTRUMENTATION AND METHODOLOGY: Texture Studies on Edible Protein Fibres Produced By A Wet Spinning Technique. I. Fibres Produced From Casein and Carrageenan. G. Downey* and K. J. Burgess**.INSTRUMENTATION AND METHODOLOGY: Texture Studies on Edible Protein Fibres Produced by a Wet Spinning Technique. 11. Fibres Produced From Casein and Alginate. G. Downey* and K. J. Burgess**.INSTRUMENTATION AND METHODOLOGY: Mechanism of Gel Formation By Low Methoxyl Pectins. R. A. Padival, S. Ranganna and S. P. Manjrekar.INSTRUMENTATION AND METHODOLOGY: A Comparison of the Texture of Expanded Milk Protein Extenders and TVP. J. J. Tuohy, K. J. Burgess and L. Lambert.INSTRUMENTATION AND METHODOLOGY: The Use, o f hlutton Tallow Fat in Fish Sausages. H. Turgut, D. Pearson and J. Rwnnan.INSTRUMENTATION AND METHODOLOGY: Assessment of Polysaccharides as Ice Cream Stabilisers. John I. L. Cottrell, G. Pass and G. 0. Phillips.

The 10% silver nitrate spray: an alternate local treatment of burns

Between May 1977 and July 1978, 307 burn patients were treated by a 10% silver nitrate spray at the Surgical Department of the Cerrahpasa Medical Faculty in Istanbul. The silver proteinate film which forms a black crust on the burnt skin protects the wound from infection through its bactericidal effectiveness and also from loss of fluid, protein, electrolytes and bodyheat. The silver film method was applied in 205 outpatients and in 102 hospitalized patients (70 children, 32 adults). The mortality rate of this data group was 7% but no renal, hepatic or hematological toxic effects due to the silver nitrate were demonstrated by the Perkin-Elmer 300 Atomic Absorption Spectrophotometer, and in the early as in the late controls of blood samples and skin biopsies no argyria was seen.

Generalized argyria secondary to chewing photographic film: Report of a case

Systemic argyria can result from the ingestion of silver salts or silver protein colloids. A case of systemic argyria that has persisted for 16 years secondary to chewing of photographic film is reviewed.

Epithelial Changes in or of the Middle Ear Mucoperiosteum

In this study the time necessary for the increase of the secretory cells in the middle ear mucosa was investigated by EM and SEM and by histochemical observation of the secretory cells. The first experiment revealed that a minimum of six to seven days was required for the renewal of the columnar cytoarchitecture after the exposure to silver protein solution. In the next experiment 35 guinea pigs were subjected to tubal obstruction. Columnar metaplasia was found in one case sacrificed after the third month, and gland formations without myoepithelial cells and nerve endings were seen in those after the fourth month. In an additional experiment, since the frontal sinus of the dogs is simple and pneumatized, duct obstruction was performed. Slight epithelial change and accumulation of glue-like thick mucus, which was negative in culture, was found in all four cases after three months. In one of the two cases with obstruction for four months, typical exophthalmus in the pseduostratified epithelium with an increase in secretory cells or cuboidal epithelium with nonciliated cells was demonstrated.

The fine structure of the stomach mucosa of the llama (Llama guanacoe)

The epithelium of the fundic region mucosa of the hind stomach in the Llama guanacoe has been studied using morphological and histochemical methods. Morphology suggests that solute and water absorption may occur in the epithelium of the surface and of the foveolae, although this absorption can not be estimated because of the extensive secretion of the gastric glands. The same cells of the surface and foveolar epithelium show numerous secretory granules. The glands reveal neck cells, chief cells, a large number of oxyntic cells, four types of endocrine cells (A-like, ECL, D and EC), brush cells and wandering cells. PAS and Alcian blue reactions for light microscopy suggest a secretion of neutral and acidic mucosubstances in the surface and foveolar epithelium, of neutral mucosubstances only in the neck cells. Periodic acid-thiocarbohydrazide silver proteinate (PA-TCH-SP) reaction for electron microscopy confirms the presence of neutral mucosubstances within the secretory granules of the surface, foveolar and neck epithelial cells. In all these cells, the reaction product is also evident within sacculi and vesicles of the maturing surface of the Golgi apparatus. A positive PA-TCH-SP reaction also occurs on the membrane (and not on the contents) of the Golgi apparatus (maturing surface) and of the secretory granules of the chief cells as well as on the membrane of the Golgi apparatus and of apical vesicles and tubules of the oxyntic cells. In addition, silver granules slightly enhance the electron desity of the contents of the secretory granules in the endocrine cells. Morphological and histochemical findings are discussed and compared with results described by others for monogastric mammals.

2(1H)-quinolinethione derivatives as organic reagents. II. Properties of some 2(1H)-quinolinethione derivatives and application to the potentiometric titration of the silver ion.

Halogen derivatives of 2 (1H)-quinolinethione, alkyl derivatives of 1, 2-dihydro-2-thioxo-4-quinolinecarboxylic acid, and alkyl 1, 2-dihydro-2-thioxo-4-quinolinecarboxylate were synthesized. The detection limits for metal ions with these reagents were examined by spot tests. The metal ions which gave a precipitate with these reagents were the same as those reported previously. iso-Amyl 1, 2-dihydro-2-thioxo-4-quinolinecarboxylate (ATQ) forms a 1-to-1 compound with silver, and an ethanolic solution of ATQ was used as a titrant to determine 0.01-0.0001 M silver ions potentiometrically. The coefficient of variation was 1.8% (n=10) for 107.4 μg of silver. Silver protein was titrated potentiometrically with ATQ in 50% (v/v) ethanolic solution. The silver content was 8.0%, with a coefficient of variation of 1.8% (n=5).

Analytical studies on pyrimidine derivatives. V. Simple and rapid spectrophotometric determination of silver (I) with 5-p- dimethylaminobenzylidene-2-thiobarbituric acid.

The spectrophotometric determination of silver (I) with 5-p-dimethylaminobenzylidene-2-thiobarbituric acid (DABTB) was studied and two simple and rapid methods are proposed. One is based on photometry of the DABTB-Ag (I) complex in ethanol-buffer (pH 6) solution (method A), and the other is based on measurement of the decrease in absorbance of DABTB due to the complex formation (method B). Beer's law holds over the range of 0.04-2 μg/ml of silver (I) at 400 nm in method A, and 0-35 μg of silver (I) at 484 nm in method B. The molar extinction coefficient of the complex is 2.5×104·1·mol-1·cm-1 in method A. Hg (I, II), Au (III), Pd (II), Pt (IV), and various anions such as Br-, Cl-, I-, SCN-, CN-, S2O32-, and S2- interfered with the determination. These methods were successfully applied to the determination of silver (I) in commercial preparations such as silver nitrate eye lotion and silver protein.

A simple impregnation technique for thin and semithin enterochromaffin cells in sections.

Constant, intense and precise impregnation of enterochromaffin (EC) cells was achieved simply by floating thin or semithin sections of gut mucosa, fixed in osmium tetroxide or in glutaraldehyde with postfixation in osmium, on a silver nitrate or proteinate solution. EC cells alone showed impregnation in the light microscope. In the electron microscope, impregnation affected not only the secretory granules of EC cells but also, although much more faintly, those of other, non-EC cells (D, X, D1, G and other cells). Lysosomes also showed partial or total reactivity. Oxidation reduced but did not entirely suppress EC cell staining and had no effect on non-EC endocrine cell staining. Since the reaction did not occur with glutaraldehyde alone, osmium appeared to be a crucial component of the process. These findings should be borne in mind in applying Thiery's method for vicinal glycol groups to the type of study material used in these experiments.

クルマエビの食道上神経節にみられるPAS陽性顆粒含有細胞の電顕的観察

As reported previously, peculiar cells were detected in the supraoesophageal ganglion of the prawn. These cells contained a granular substance, resernbling a grain of rice in shape. Histochemical exmination under light microscope revealed that the substance was PAS-positive. The present study by electron microscopy deals with the ultrastructure and the re-identification of the cytochemical character of this substance. The results obtained from this study are as follows. 1) The granular substance possesses no limiting membrane and seems to have a polysaccharide character, because of positive reaction to silver proteinate. 2) Internally and peripherally, there exist many cored or granular vesicles, which are suggested to be functionally associated with Golgiapparatus.