Application of Phase-Contrast Microscopy to Schiff-Positive Material

It was observed that ground substance between the smooth muscle fibers in cerebral arteries stained by periodic acid-Schiff (PAS) was red as seen by ordinary bright-field microscopy (BF), but blue as observed by phase-contrast microscopy (PC). The basement membranes in the small intestine and around the kidney tubules, as well as the striated borders of the intestinal epithelium and the brush borders of the kidney tubules, were seen in blue when stained by PAS and observed by PC. The cytoplasm of PAS stained liver cells, when observed by PC, had irregular shaped areas of blue interspersed between the red material. This blue color was seen by PC after PAS, ninhydrin-Schiff and the Feulgen procedures. Our evidence suggests that this phenomena is characteristic of Schiff-positive material. Digestion by various enzymes: malt diastase, testicular hyaluronidase, collagenase, pepsin, pectinase, trypsin and DNase showed different effects on ground substance, liver cells, basement membranes, and brush and striated borders.     

Histological and carbohydrate histochemical studies of the nasal mucosa of sheep in Saudi Arabia with special reference to its glandular tissue

The histology and carbohydrate histochemistry of the nasal mucosa with attention to glandular tissue had been studied in 7 heads of sheep. Tissues were taken from vestibular region, septum at level of the alar fold, rostral portion of nasal conchae, caudal portion of nasal conchae, middle portion of septum and ethmoidal conchae region. Stratified squamous nonkeratinized epithelium was observed covering the vestibular region. The propria-submucosa of the nasal vestibule was richly permeated with glands having affinity for PAS and non-alcianophilic. The post-vestibular portion of the nasal cavity was lined by transitional epithelium and caudal to it, stratified columnar nonciliated epithelium was noticed. The respiratory epithelium covered the caudal half of the nasal conchae and the major portion of the septum as well as the recesses of the ethmoidal conchae. The glands associated with the respiratory mucosa were thick, coiled and tubular, containing both nonalcianophilic PAS positive and alcianophilic PAS positive cells. The olfactory mucosa covered the ethmoidal conchae and showed predominant serous glands. The results were discussed with that given for other mammals and in regard to the respiratory functions of the nasal mucosa.     

Isolation of sperm bindin from the oyster (Crassostrea gigas)

A method was devised for isolating the insoluble content of the acrosome granule of sperm of the oyster Crassostrea gigas. The method involves the dissolution of the entire cell, except for the acrosome granule, in the detergent sodium lauroyl sarcosinate (sarcosy I). The isolated acrosome granule content is ring-shaped and is 84% protein by weight. SDS-polyacrylamide gel electro-phoresis of this material yields from 1 to 4 bands of 65,000; 53,000; 43,000 and 34,000 apparent molecular weight, all of which stain with the PAS reaction indicating the material is a glycoprotein. The 65,000 molecular weight component is always present, but the presence of the other three bands varies with each preparation. The isolated acrosome granules agglutinate formaldehyde-fixed oyster eggs. A trypsin-generated glycopeptide digest of oyster egg surfaces inhibits the agglutinin activity of the isolated acrosome granules. We propose that the acrosomal glyco-protein material is oyster sperm bindin which functions as the adhesive substance bonding the sperm to the egg during fertilizaion.     

A PERIODIC ACID-SCHIFF'S SUBSTANCE RELATED TO THE DIRECTIONAL GROWTH OF POLLEN TUBE INTO EMBRYO SAC IN PASPALUM OVULES

The presence of a periodic acid-Schiff's (PAS) substance at the micropylar portion of the ovules of Paspalum orbiculare and three other species of Paspalum has been revealed by improved cytological techniques. The substance is located in the distal portion of the outer integument, especially at its edges, and in the micropyle, which is formed by the inner integument. A study of the origin of this substance in P. orbiculare indicates that it is produced from the cellular material of the dissolved cells in situ. The PAS substance is highly water soluble and may contain some protein. The final directional growth of the pollen tube into the filiform apparatus of the degenerated synergid is related to this substance. The role of the synergids in its formation is discussed.     

A structural investigation of the ovule in sugar beet, Beta vulgaris: Integuments and micropyle

The micropyle and the integuments of sugar beet (Beta vulgaris) ovules have been investigated by light and electron microscopy during differentiation and maturation of the ovule. The micropyle itself is formed by the inner integument which is surrounded by the outer integument at its base. The micropyle containts a fibrillar PAS+ substance and is often covered by a thin sheet or hymen. Both integuments are cuticle-covered thin sheets, each 2-few cell layers in thickness. In the outer integument an increase in starch accumulation occurs during ovule maturation and probably functions as nutrient storage for embryo development. The inner epidermis of the inner integument differentiates as the most conspicuous cell layer of the beet ovule. During growth and maturation of the ovule a system of small perinuclear vacuoles containing dense material increases steadily in these cells. At maturity this system fills up more than half of each cell and very dense material has accumulated in each vacuole. This vacuole content is highly refractive and contains tannins and/or polyphenols.     

Histochemical demonstration of acid mucopolysaccharide production by tissue cultured epithelial-like cells

Summary The production of mucopolysaccharides by human epithelial-like cells in vitro has been studied using histochemical techniques. Cells of the human amnion F/L line were grown for 12–14 days in Leighton tubes containing cover slips which were fixed and stained by the Hale, PAS, toluidine blue and alcian blue stains and techniques. Control slides were treated with active and heat inactivated solutions of testicular and bacterial hyaluronidase and chondroitinase prior to staining. Results indicated that material stainable by all the techniques employed was elaborated by the amnion cells and this was partially removable by various combinations of polysaccharases. It is concluded that epithelial-like cells can synthesize and elaborate the ground substance components, hyaluronic acid, chondroitin sulphates and unidentified polysaccharide complexes, in much the same manner as mesenchymal cells and thus contribute to the maintenance of epithelial integrity by de novo production of intercellular substances.Supported by USPHS Grants No. 991 and DE-01681, T01-DE00108 National Institute of Dental Research, National Institutes of Health and the National Health and Medical Research Council, Australia.     

Sex-specific glycoproteins in Chlamydomonas flagella an immunological study

To identify mating type-specific glycoproteins associated with the flagellar membrane of Chlamydomonas eugametos, which could be involved in sexual agglutination, antibodies were raised in rabbits against purified gamete flagella of either mating type. The immunoglobulin (Ig) fractions exhibited partial mating-type specificity in agglutinating gametes, in the indirect immunofluorescence test and in the crossed immunoelectrophoresis test. This specificity was strongly enhanced by absorbing the fractions with flagella of the opposite mating type. Absorbed Ig fractions produced a single precipitation line with Triton extracts of gamete flagella in the crossed immunoelectrophoresis technique. On polyacrylamide gel electrophoresis this line appeared to contain two flagellar glycoprotein fractions, PAS 1 and PAS 4. Polyacrylamide gels of flagellar extracts incubated with these Ig fractions, followed by staining with peroxidase-anti-rabbit Ig resulted in the staining of only the PAS 1 and PAS 4 bands, which confirms that these components of the flagellar membrane are mating type-specific antigens.The investigations were supported by the Foundation for Fundamental Biological Research (BION), which is subsidized by the Netherlands Organization for the Advancement of Pure Research (ZWO).     

Oviducal gland microstructure of Raja miraletus and Dipturus oxyrinchus (Elasmobranchii,Rajidae)

We studied the morphology and histology of the oviducal gland (OG) in the brown ray (Raja miraletus) and the long‐nosed skate (Dipturus oxyrinchus) to understand its functional role in the reproductive strategy of these species. The external morphology of the gland was similar in both species, with lateral extensions like those found in other members of the Rajidae. Microscopic analysis showed a similar internal organization in both species. Immature and developing glands did not react to histochemical techniques. On reaching maturity, the OG had the largest width due to an increase in the production of secretory materials. In both species, the club zone of the gland showed a strong reaction to Periodic acid‐Schiff (PAS) and alcian blue (AB) stains, indicating production of neutral and sulfated acid mucins. The secretory material produced by the papillary zone varied greatly between the two species. Both displayed tubular glands similar to those observed in the club zone, but in D. oxyrinchus the region near the lumen was intensely PAS+, whereas the last row of tubules of the brown ray stained intensely for a mixture of neutral and sulfated mucins. The baffle zone was the most conspicuous and extensive segment of all OGs, and it did not react to PAS/AB. The terminal zone, which is responsible for production of hair filaments, differed between the two species in terms of composition and organization of serous and mucous glands. This difference probably is related to the different substrates in which they release the egg capsules. Individual sperm detected in the brown ray baffle lamellae could be the result of a recent mating, whereas their presence in the deep recesses of the baffle and in the terminal zone of the long‐nosed skate might indicate sperm storage. J. Morphol. 276:1392–1403, 2015. © 2015 Wiley Periodicals, Inc.     

THE SURFACE FEATURES OF DROSOPHILA EMBRYONIC CELL LINES

Schneider's cell lines 1, 2 and 3 derived from embryonic stages of Drosophila melanogaster were examined by scanning electron microscopy, histochemical staining procedures, and SDS polyacrylamide disc gel electrophoresis. Although the three lines showed some similarities when compared by each of these methods, differences between the lines were observed as well. The surface features of the cells in each line showed morphologic as well as age dependent distinctions when examined in the scanning electron microscope. The intracellular distribution and amount of periodic acid-Schiff positive (PAS+), α-amylase sensitive material was also distinct for each of the lines. Approximately one-third of line 1 cells contained PAS+ hyaluronidase-sensitive material localized within a cytoplasmic vesicle whereas less than 1% of the cells of lines 2 and 3 contained such material. SDS polyacrylamide disc gel electrophoresis revealed PAS+ material in a soluble fraction of line 2 cells and in the 100,000 μ g pellet of line 3 cells, but no PAS+ bands in similar fractions of line 1 cells were detected.     

An interesting case of colloidal iron positivity

Summary An interesting case of a colloidal iron (CI) positive basophilic substance in the adrenal medullary cells of amphibia and reptilia is reported here. The substance, however, does not stain by alcian blue (AB). It is negative to PAS, Azure A, aldehyde fuchsin, AB at pH 1 and MgCl₂ — AB though orthochromatically stained by toluidine blue at pH 3. More work is needed to establish the exact nature of the CI positive material.     

Extracellular compartments in matrix morphogenesis: collagen fibril, bundle, and lamellar formation by corneal fibroblasts

The regulation of collagen fibril, bundle, and lamella formation by the corneal fibroblasts, as well as the organization of these elements into an orthogonal stroma, was studied by transmission electron microscopy and high voltage electron microscopy. Transmission and high voltage electron microscopy of chick embryo corneas each demonstrated a series of unique extracellular compartments. Collagen fibrillogenesis occurred within small surface recesses. These small recesses usually contained between 5 and 12 collagen fibrils with typically mature diameters and constant intrafibrillar spacing. The lateral fusion of the recesses resulted in larger recesses and consequent formation of prominent cell surface foldings. Within these surface foldings, bundles that contained 50-100 collagen fibrils were formed. The surface foldings continued to fuse and the cell surface retracted, forming large surface-associated compartments in which bundles coalesced to form lamellae. High voltage electron microscopy of 0.5 micron sections cut parallel to the corneal surface revealed that the corneal fibroblasts and their processes had two major axes at approximately right angles to one another. The surface compartments involved in the production of the corneal stroma were aligned along the fibroblast axes and the orthogonality of the cell was in register with that of the extracellular matrix. In this manner, corneal fibroblasts formed collagen fibrils, bundles, and lamellae within a controlled environment and thereby determined the architecture of the corneal stroma by the configuration of the cell and its associated compartments.     

Studies on the electroretinogram. II. Model of an a-wave regenerator mechanism

A model is proposed to relate the regeneration of the ERGa-wave after partial light adaptation to the level of the light adaptation. The model assumes that thea-wave amplitude is a function of some reactive substance associated with ana-wave generator. The maximuma-wave amplitude occurs when the eye is fully dark adapted, and thea-wave generator initiator concentration is at a maximum. Thea-wave generator initiator concentration can be decreased by interacting with a product of the rhodopsin-light energy reaction, and increased by removal of this inhibitor. The removal of the inhibitor depends upon the isomerization of the all-trans-retinene to the 11-cis form. An excess of inhibitory material overa-wave generator initiator would cause a delay in the appearance of thea-wave until the excess inhibitory material is removed. This delay is a linear function of the logarithm of the adapting energy. The agreement of this model with the experimental ERG data is very good. Supported in whole by Public Health Service Research Grant No. NB-02522, from the National Institute of Neurological Diseases and Blindness, Bethesda, Maryland.     

The demonstration of plasmalogen with periodic acid

Summary The same distal convolutions in the outer cortex of the kidney of the rat reacted after either the PAS or the plasmal reaction. This was substantiated by comparing PAS-positive structures in an unfixed, frozen section with plasmal-positive structures in an adjacent section. The absence of PAS-positive material after aldehyde blockade and lipid extraction procedures indicated that the PAS-positive material was lipid in nature and was probably plasmalogen. Cortical PAS-positive material was only observed in the epithelium of the distal tubules and these were the only tubules in the cortex containing plasmalogen. This was cited as empirical evidence for periodic acid demonstration of plasmalogen in particular and not unsaturated lipids in general. It is strongly suggested that controls insuring the absence of plasmalogen-derived aldehydes should be prepared by extracting unfixed, frozen sections with chloroform-methanol or by treating them with acetic acid-aniline before carrying out the PAS reaction.This investigation was supported by USPHS Grant GM-407 from the National Institutes of Health and by University of Washington Graduate School Research Funds, Initiative 171.A portion of this work was carried out in the Department of Anatomy of the State University of New York at Buffalo, Buffalo, New York.     

Effectiveness of aroma on work efficiency: lavender aroma during recesses prevents deterioration of work performance

The present study investigated whether exposure to aromas during recess periods affects work performance. Subjects comprised 36 healthy male students (mean age, 24.2 +/- 2.2 years) who were randomly divided into three groups: (1) control group, not exposed to aroma during recesses; (2) jasmine group, exposed to jasmine aroma during recesses; and (3) lavender group, exposed to lavender aroma during recesses. All participants completed five work sessions performing a task requiring concentration on a computer monitor, with each session lasting 60 min. Recess periods of 30 min were provided between each session. To clarify the time at which work concentration was lowest, work performance for the control group was analyzed. Concentration was lowest in the afternoon period, where afternoon drowsiness is strongest. Comparison of the three groups for this time period indicated significantly higher concentration levels for the lavender group than for the control group. No such effect was noted for the jasmine group. Although lavender is a sedative-type aroma, use during recess periods after accumulation of fatigue seems to prevent deterioration of performance in subsequent work sessions.     

A puzzling feature of Colloidal Iron positive and Alcian Blue negative material

Summary A puzzling feature of Colloidal Iron positive and Alcian Blue negative substance is encountered in yolk sac of young larvae of a fish —Tilapia mossambica. This yolk material is PAS positive (proved to be due to neutral mucopolysaccharide) and negative to Toluidine Blue, Azure A (pH 2 to 4.5), Aldehyde Fuchsin and AB pH 1. More work is necessary to establish the exact chemical nature of the CI positive material.     

Biomorphosis of the human fallopian tube mucosa; a contribution to the problem of morphology and aging of the uterine tube. II. Histochemical findings

The present histochemical findings of investigations on the biomorphosis of the human tubal mucosa indicate that mucosubstances and glycogen localized in the epithelium exhibit age-dependent changes with regard to occurrence and localization. In the embryo-fetal time PAS-positive, diastase-resistant substances are localized in the epithelium, at first basally, and later perinuclearly. In the neonatal phase the distal tubal epithelium has only a weak PAS reaction, and the proximal epithelium has a detectable supranuclear activity. In the end of the 1st decade of the life the epithelium possesses a periodate reactive diastase-sensitive material densely deposited in the preampullar and ampullar parts of the uterine tube, preferably. Afterwards PAS-positive diastase-sensitive and diastase-resistant substances, respectively, are regularly present, in which in the fertile age of the women a regular pattern of the PAS activity can be demonstrated. In the period of the regressive age it is possible to establish a increasing disturbance of the usual cellular picture of the tubal epithelium. In connection with the structural changes a increase of histochemical different reacting cell groups is evident. As a result, a dissociated cellular picture has developed. Epithelial glycoproteins and glycogen can be detected in the mucosa up to the phase of the senium.     

The fine structure of the subcommissural cells and of Reissner's fibre in Myxine

Summary Reissner's fibre and the subcommisssural cells mainly from Myxine were investigated by light and electron microscope methods. The subcommissural cells carry several cilia and produce a chrome haematoxyphile secretion in the form of granules. It is probable that the nucleus as well as the mitochondria are involved in the synthesis of this material. The secretory release suggests an apocrine type in which the granules swell and form a fine-granulated, chrome haematoxyphile substance in the ventricle. Caudally in the subcommissural canal this material condenses to form Reissner's fibre, which in electron micrographs, except for a fine-granulated ground substance, does not show any structures, a limiting membrane or traces of cell organelles or remnants.Aided by grants from the Swedish Natural Science Research Council.     

Interrupted incubation: How dabbling ducks respond when flushed from the nest

Nesting birds must provide a thermal environment sufficient for egg development while also meeting self‐maintenance needs. Many birds, particularly those with uniparental incubation, achieve this balance through periodic incubation recesses, during which foraging and other self‐maintenance activities can occur. However, incubating birds may experience disturbances such as predator or human activity which interrupt natural incubation patterns by compelling them to leave the nest. We characterized incubating mallard Anas platyrhynchos and gadwall Mareca strepera hens’ responses when flushed by predators and investigators in Suisun Marsh, California, USA. Diurnal incubation recesses initiated by investigators approaching nests were 63% longer than natural diurnal incubation recesses initiated by the hen (geometric mean: 226.77 min versus 142.04 min). Nocturnal incubation recesses, many of which were likely the result of predators flushing hens, were of similar duration regardless of whether the nest was partially depredated during the event (115.33 [101.01;131.68] minutes) or not (119.62 [111.96;127.82] minutes), yet were 16% shorter than natural diurnal incubation recesses. Hens moved further from the nest during natural diurnal recesses or investigator‐initiated recesses than during nocturnal recesses, and the proportion of hen locations recorded in wetland versus upland habitat during recesses varied with recess type (model‐predicted means: natural diurnal recess 0.77; investigator‐initiated recess 0.82; nocturnal recess 0.31). Hens were more likely to take a natural recess following an investigator‐initiated recess earlier that same day than following a natural recess earlier that same day, and natural recesses that followed an investigator‐initiated recess were longer than natural recesses that followed an earlier natural recess, suggesting that hens may not fulfill all of their physiological needs during investigator‐initiated recesses. We found no evidence that the duration of investigator‐initiated recesses was influenced by repeated visits to the nest, whether by predators or by investigators, and trapping and handling the hen did not affect investigator‐initiated recess duration unless the hen was also fitted with a backpack‐harness style GPS–GSM transmitter at the time of capture. Hens that were captured and fitted with GPS–GSM transmitters took recesses that were 26% longer than recesses during which a hen was captured but a GPS–GSM transmitter was not attached. Incubation interruptions had measurable but limited and specific effects on hen behavior.     

Isolation,and biochemical and biological characterization of an a-mating-type-specific glycoprotein responsible for sexual agglutination from the cytoplasm of a-cells,in the yeast Saccharomyces cerevisiae

An a-mating-type-specific substance responsible for sexual agglutination was purified to 397-times in specific activity (units/mg protein) from the cytoplasm of a-mating type cells. The purified substance gave a single band stained with PAS reagent but not with both Coomassie brilliant blue and silver staining reagent by polyacrylamide gel electrophoresis in the presence of 8 M urea. However, incorporation of [³⁵S]methionine and Lowry reaction clearly indicate that the substance is a glycoprotein. The substance specifically masked sexual agglutinability of cells of the opposite mating type , indicating univalent action. The substance is a glycoprotein with a carbohydrate content of 90%, a pI of 4.5, and a molecular weight of 130,000. The substance was inactivated by 2-mercaptoethanol and proteolytic enzymes but not by glycolytic enzymes. The substance formed a complementary complex having no biological activity when mixed with -agglutination substance from the wall or cytoplasm of -cells in vitro.Non-common abbreviations PAGE polyacrylamide gel electrophoresis - PAS periodic acid-Schiff - PBS 10^-2 M phosphate buffer solution, pH 5.5 - PMSF phenylmethyl sulfonyl fluoride - SDS sodium dodecyl sulfate     

The periodic acid-schiff reaction in neutrophil leukocytes-influence of fixation and amylase digestion

Summary The periodic acid-Schiff (PAS) reaction in normal human neutrophil leukocytes was studied morphologically and microspectrophotonietrically after different fixation methods and after treatment with -amylase and -amylase.The best fixation methods with respect to the morphological distribution of the stain and to the quantitatively measured amount of PAS positive material preserved in the cells were fixation with absolute methanol, absolute ethanol, acetic alcohol formalin and Rossman's fluid as well as fixation by freeze substitution. The amount of PAS reactive material in the cells after these fixation methods was in the same range as in unfixed cells covered with a semi-permeable formvar membrane. The PAS positive material could be removed by treatment with -amylase after most fixatives, the most important exception being acetic alcohol formalin, after which fixative no reduction was obtained in the amount of PAS positive material. This may be due to the fixative creating an -amylase resistant protein-glycogen binding, -amylase digestion gave only a slight reduction in the amount of PAS reactive material.The results indicate that the main part of the PAS reactive material in normal human neutrophil leukocytes consists of glycogen. It is probably preserved to about 90 per cent or more by the above mentioned fixatives.

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Zusammenfassung Die Perjodsäure-Schiff-(PAS-)Reaktion in normalen menschlichen neutrophilen Leukocyten wurde morphologisch und mikrospektrophotometrisoh nach verschiedenen Fixierungen und nach - und -Amlyase-Behandlung untersucht.Die besten Fixierungsmittel waren — in Hinsicht auf die Verteilung und die quantitativ bestimmbare Menge der erhaltenen PAS-positiven Substanz — absolutes Methanol, absolutes Äthanol, acetic-alcohol-formalin und Rossmans Fixierungsmittel. Fixierung mittels Gefrier-Substitution hat sich ebenfalls bewährt.Die Menge der nachweisbaren PAS-positiven Substanz lag nach diesen Vorbehandlungen in der gleichen Größenordnung, wie bei nicht fixierten, mit einer semipermeablen Formvarmembran überzogenen Zellen.Die PAS-positive Substanz war durch -Amylase-Behandlung nach den meisten Fixierungen entfernbar; die wesentliche Ausnahme bildet acetic-alcohol-formalin, hiernach war keine Entfernung der PAS-positiven Substanz möglich. Die Ursache wird in einer -Amylase-resistenten Protein-Glykogen-Bindung gesehen. -Amylase führte zu einer nur geringfügigen Abnahme PAS-positiven Materials.Die Ergebnisse zeigen, daß der größere Teil des PAS-positiven Materials in normalen menschlichen Leukocyten Glykogen ist. Es wird mindestens zu 90% durch die oben erwähnten Methoden fixiert.

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This work has been supported by grants from the Swedish Medical Research Council and the Wallenberg Foundation.

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The Fibrinogen was kindly supplied by the Kabi AB, Stockholm, Sweden.