The fine structure of the glycocalyx of equine spermatozoa: a high-resolution cytochemical study.

Equine spermatozoa were obtained from ejaculates of young stallions. The seminal plasma was removed and the sperm pellets washed three times with 0-15 M-NaCl solution before final centrifugation at 4500 g for 15 min. The pellets were fixed in a mixture of 2-5% glutaraldehyde in 0-1 M-cacodylate buffer, pH 7-4, with 0-5% Alcian blue and post-fixed in 1% osmium tetroxide with 1% lanthanum nitrate; other samples were treated with ruthenium red. All samples were dehydrated in ascending concentrations of ethanol, embedded in araldite and thin sections examined in an electron microscope. Electron dense deposits of lanthanum were present on the surface plasmalemma of the head, mid-piece and tail of 70% of mature spermatozoa, and similar deposits were seen in ruthenium red-treated samples. No glycocalyx was observed in untreated spermatozoa.     

Isolation of transverse tubules by fractionation of sarcoplasmic reticulum preparations in ion-free sucrose density gradients

A new method for the preparation of transverse tubules (T-tubules) from rabbit skeletal muscles is reported. When crude sarcoplasmic reticulum (SR) preparations were centrifuged on sucrose density gradients containing buffering ions (buffered gradients) 70-80% of the material sedimented as a single heavy band in the region of 43% sucrose. When this fraction (or crude SR) was recentrifuged on sucrose gradients prepared free of buffer or other ions (ion-free gradients) the heavy band dissociated into three fractions of different densities. The lightest fraction sedimented at 28% sucrose and was identified as T-tubules on the basis of its nitrendipine and ouabain binding properties. The enzymatic properties, cholesterol contents, and protein compositions of the fractions obtained when SR is centrifuged on buffered and ion-free sucrose density gradients were measured. The T-tubules were enriched in cholesterol and in marker enzymes for surface membranes while the other fractions were shown to be terminal cisternae and longitudinal cisternae on the basis of their (Ca2+,Mg2+)-ATPase activities and characteristic protein profiles.     

The Separation and Isolation of Plasma Membranes and Mesosomal Vesicles from Staphylococcus Aureus

We have separated and Isolated the plasma membranes and mesosomal vesicles of Staphylococcus aureus ATCC 6538P. Cells were grown aerobically in Difco synthetic AOAC broth, washed and resuspended in hypertonic buffer (3.45 M NaC1) containing 0.02 M MgSO₄. Cell wall was removed by treatment with lytic enzyme from S. aureus, strain LS. The protoplasts were collected by centrifugation at 10,000 × g for 1 hour, resuspended in hypotonic buffer containing 0.02 M MgSO₄ and lysed. The resultant plasma membranes were washed and centrifuged on a 60tr>75Z sucrose density gradient at 55,000 × g for 15 hours. Gradient patterns showed two bands of membranes. Crude mesosomes were obtained from the 10,000 × g supernatant fractions by centrifugation at 100,000 × g for 2 hours. The reddish-brown gelatinous pellet, which consisted of mesosomal vesicles and a few ribosomes, was washed and centrifuged on a 60 to 85% sucrose density gradient at 100,000 × g for 15 hours. Gradient patterns produced two bands of mesosomal vesicles. The homogeneity of the plasma membranes and mesosomal vesicles was determined by electron microscopy and chemical analyses.     

Oxidation of ruthenium red for use as an intercellular tracer

Summary When ruthenium red (RR) is combined with OsO₄, an electronopaque complex forms which readily binds to the cell surface coat. However, the RR-OsO₄ complex is often excluded from intercellular spaces in many cell types, and thus is not dependable as a tracer of regions continuous with the extracellular space. Postfixation of erythrocytes agglutinated by the lectin helix (Helix promatia) and intact carotid artery endothelium with a freshly prepared mixture of 1% OsO₄ containing 0.1% ruthenium red (RR) resulted in a dense surface deposit of these cells, but intercellular regions were penetrated to a minimal degree by the stain. When a similar mixture of RR-OsO₄ was allowed to stand 3 h before use, RR is oxidized by OsO₄ to yield a ruthenium compound that has a spectrophotometric absorbance maximum at 365 nm. This RR molecule has a reduced number of cationic sites due to binding with osmium dioxide OsO ₂ ⁼ . Postfixation of agglutinated RBCs and carotid artery endothelium with this oxidized ruthenium-OsO₄ mixture resulted in a 50–80% decrease in surface deposition but markedly enhanced penetration into intercellular regions. The enhanced penetration is attributed to decreased binding affinity of the oxidized ruthenium for anionic surface membrane components, permitting effective stain penetration into regions of cell-to-cell contact rather than extensive surface deposition. These studies indicate that the ruthenium compound formed by OsO₄ oxidation of ruthenium red may be a useful tracer for ultrastructural visualization of intercellular spaces and junctions.     

Chloroplast Integrity and ATP-Dependent CO(2) Fixation in Spinacia oleracea

Washed whole chloroplasts of Spinacia oleracea isolated and assayed in a tris (hydroxymethyl aminomethane)-HCl buffered sucrose solution exhibited low dark CO₂ fixing activity, whereas washed whole chloroplasts isolated in the same buffer but assayed in that buffer without sucrose exhibited much greater dark CO₂ fixing activity. The lowered activity could be attributed to the impermeability of the chloroplast membrane to ribose-5-phosphate or adenosine triphosphate. The preservation of the integrity of the chloroplast membrane, as reflected by its impermeability to either or both of the abovementioned compounds, was measured by the fixation of ¹⁴CO₂ into acid-stable products in the presence of ribose-5-phosphate and adenosine triphosphate by the whole chloroplast as compared with fixation by the chloroplast extract. An effect (i.e., apparent resistance to the passage of ribose-5-phosphate or adenosine-5-triphosphate into the chloroplast) similar to, but less pronounced than, that produced by the presence of sucrose in the isolation medium was observed upon the addition of MnCl₂ or CaCl₂ to the buffered sucrose isolation medium. The addition of KCl enhanced slightly the effect produced by addition of sucrose alone to the isolation medium. The presence of MgCl₂ in the isolation medium, however, either caused the chloroplasts to become leaky or more fragile since more of the activity of the carboxylative phase enzymes appeared in the cytoplasm. When a mixture of all of the metal ions was added to the buffered sucrose suspending medium, the chloroplasts exhibited the same response observed with MgCl₂ alone. The addition of ethylene diaminetetraacetate or dithiothreitol appeared to alter the permeability of the chloroplast membrane nonspecifically when the assay was conducted in the absence of sucrose. Specific activities (μmoles CO₂ fixed/mg chlorophyll × hr) as high as 329.6 have been observed for dark fixation by chloroplasts. The phosphoenolpyruvate carboxylase activity in the chloroplasts was only one-seventh that of ribulose diphosphate carboxylase. The phosphoenolpyruvate carboxylase activity in the cytoplasm was 5 times that of the chloroplasts.     

The adverse effects of HEPES,TES, and BES zwitterion buffers on the ultrastructure of cultured chick embryo epiphyseal chondrocytes

Summary Chick embryo epiphyseal chondrocytes cultured in media containing HEPES, TES, and BES zwitterion buffers, used in combination or independently, consistently developed cytoplasmic vacuoles. This cytoplasmic vacuolation was resolved when the zwitterion buffered media was replaced by media containing bicarbonate:CO₂ enriched air buffer. Vacuoles were infrequent or absent in cultures grown in bicarbonate:CO₂ enriched air. Chondrocytes with an established extracellular matrix showed less vacuolation than fibroblastlike and polygonal shaped cells that lacked such a matrix. The granular endoplasmic reticulum and Golgi dictyosomes of zwitterion buffered chondrocytes were distended and contained a flocculent amorphous material. Cytoplasmic vacuoles (0.5 to 3.0 μm diam) formed by the fusion and intracellular accumulation of Golgi vesicles and vacuoles also contained a flocculent material enhanced by ruthenium red. Membrane bound extracellular vacuoles containing ruthenium red stained proteoglycan aggregates were common in the extracellular matrix of zwitterion buffered cultures but were generally absent from bicarbonate treated cultures. Electron dense calcium deposits seemed much larger and more numerous in the presence of zwitterion buffers. It is suggested that HEPES, TES, and BES buffers, used alone or in combination, may adversely affect cell membrane systems, and thus the transport or secretory mechanisms operative in cultured chondrocytes, or both, resulting in vacuole formation and the intracellular accumulation of synthesized export material. Although the mechanism by which HEPES, TES, and BES induce these changes remains unclear, the use of zwitterion buffers in biological preparations should be treated with caution. This work forms part of a project on Connective Tissue Remodelling supported and financed by the Medical Research Council of New Zealand, of which M. H. F. is a Career Fellow.     

Modifcation of the French Azure C Tissue Stain

A staining procedure for monocytes in specimens of blood and bone marrow was developed. The technique was a two step procedure in which unfixed cells were exposed first to a methanolic solution of C.I. basic blue 54. Next, an aqueous alkaline buffered solution of C.I. basic blue 141 was added to the first staining solution. After staining for 10 min in the solution with two stains, slides or coverslips were washed for 5 sec in pH 5.6 phosphate buffer and drained dry. The cytoplasm of monocytes stained intensely deep purple and frequently nuclei were stained red. Similar staining was not found in other types of normal or abnormal blood and bone marrow cells.     

Staining Raw and Cooked Apple Tissues for Study of Cell Wall Structure

Permanent preparations were made of paraffin sections from raw and cooked apple tissues stained with microchemical color reagents for pectins and pentosans. Sections stained with ruthenium red to show pectins were dehydrated and covered in balsam, and sections stained with diphenylene diamine acetate (DDA) to show pentosans were washed with water and covered in Clearcol.

Cooking was accomplished by steaming cubed histological samples. Both raw and steamed specimens were fixed in FAA in a vacuum chamber, dehydrated and cleared in tertiary butyl alcohol, and embedded in paraffin. Paraffin sections first fixed to slides with Haupt's adhesive were further stabilized by immersing in a 1% celloidin solution after dissolving the paraffin.

Ruthenium oxychloride flakes were dissolved in a Coplin jar of water containing 2 drops of ammonium hydroxide. Rehydrated sections were stained in ruthenium red 30 minutes and rinsed in water. Three methods of further preparation follow: (1) Flood sections with 10% gum arabic; drain and air-dry thoroughly; immerse in xylene 5 minutes; cover in balsam. (2) Drain and air-dry sections; if desired, counterstain dry sections with Johansen's fast green solution; immerse in xylene; cover in balsam. (3) Dehydrate by dipping in 70%, 95%, and absolute ethyl alcohol; immerse in xylene; cover in balsam.

DDA was made by heating 15 g. of benzidine in 150 ml. of glacial acetic acid and 450 ml. of water until dissolved, then adding water to make 750 ml. of solution. Rehydrated sections were stained 4 hours in DDA, washed, stained 5 minutes in Congo red (Congo red, 5 g.; NaOH, 5 g.; water, 100 ml.), washed, and covered in Clearcol.

An Autotechnicon was used for dehydration, clearing, infiltration, deparaffinizing sections, and staining. Procedures that necessarily remained manual were fixation in a vacuum chamber, and all operations that followed staining.

Ruthenium red, though the best available indicator for pectins, may not be specific for these substances. DDA and ruthenium red stained identical structures in hypodermis and cortex. DDA also stained cuticle, hence was more useful than ruthenium red for delineating that portion. DDA sections were better for photomicrography, and for measuring thickness of cell walls. Neither stain prevented the study of cell walls in polarized light.     

A Two Step Stain for Normal and Leukemic Monocytes Using Two Different Dyes Applied in Sequence

A staining procedure for monocytes in specimens of blood and bone marrow was developed. The technique was a two step procedure in which unfixed cells were exposed first to a methanolic solution of C.I. basic blue 54. Next, an aqueous alkaline buffered solution of C.I. basic blue 141 was added to the first staining solution. After staining for 10 min in the solution with two stains, slides or coverslips were washed for 5 sec in pH 5.6 phosphate buffer and drained dry. The cytoplasm of monocytes stained intensely deep purple and frequently nuclei were stained red. Similar staining was not found in other types of normal or abnormal blood and bone marrow cells.     

INFLUENCE OF GLUTARALDEHYDE AND/OR OSMIUM TETROXIDE ON CELL VOLUME, ION CONTENT, MECHANICAL STABILITY, AND MEMBRANE PERMEABILITY OF EHRLICH ASCITES TUMOR CELLS

Effects of fixation with glutaraldehyde (GA), glutaraldehyde-osmium tetroxide (GA-OsO₄), and osmium tetroxide (OsO₄) on ion and ATP content, cell volume, vital dye staining, and stability to mechanical and thermal stress were studied in Ehrlich ascites tumor cells (EATC). Among variables investigated were fixation time, fixative concentration, temperature, osmolality of the fixative agent and buffer, total osmolality of the fixative solution, osmolality of the postfixation buffer, and time of postfixation treatment in buffer (Sutherland, R. M., et al. 1967. J. Cell Physiol. 69:185.). Rapid loss of potassium, exchangeable magnesium, and ATP, and increase of vital dye uptake and electrical conductivity occurred with all fixatives studied. These changes were virtually immediate with GA-OsO₄ or OsO₄ but slower with GA (in the latter case they were dependent on fixative temperature and concentration) (Foot, N. C. 1950. In McClung's Handbook of Microscopical Technique. 3rd edition. 564.). Total fixative osmolality had a marked effect on cell volume with OsO₄ but little or no effect with GA or GA-OsO₄. Osmolality of the buffer had a marked effect on cell volume with OsO₄, whereas with GA or GA-OsO₄ it was only significant at very hypotonic buffer osmolalities. Concentration of GA had no effect on cell volume. Osmolality of the postfixation buffer had little effect on cell volume, and duration of fixation or postfixation treatment had no effect with all fixatives. Freezing and thawing or centrifugal stress (up to 100,000 g) had little or no effect on cell volume after all fixatives studied. Mechanical stress obtained by sonication showed that OsO₄ alone produced poor stabilization and that GA fixation alone produced the greatest stabilization. The results indicate that rapid membrane permeability changes of EATC follow fixative action. The results are consistent with known greater stabilizing effects of GA on model protein systems since cells were also rendered relatively stable to osmotic stress during fixation, an effect not noted with OsO₄. After fixation with GA and/or OsO₄ cells were stable to osmotic, thermal, or mechanical stress; this is inconsistent with several earlier reports that GA-fixed cells retain their osmotic properties.     

Staining Tissue of the Central Nervous System with Luxol Fast Blue and Neutral Red

The tissue is fixed in 10% neutral saline formalin for 1 day to 3 wk depending on the size of the block, dehydrated and embedded in paraffin. The sections are stained at 57° C for 2 hr, then at 22° C for 30 min, in a 0.0125% solution of Luxol fast blue in 95% alcohol acidified by 0.1% acetic acid. They are differentiated in a solution consisting of: Li₂CO₃, 5.0 gm; LiOH-H₂O, 0.01 gm; and distilled water, 1 liter at 0-1° C, followed by 70% alcohol, and then treated with 0.2% NaHSO₃. They are soaked 1 min in an acetic acid-sodium acetate buffer 0.1 N, pH 5.6, then stained with 0.03% buffered aqueous neutral red. Sections are washed in distilled water, 1 sec, then treated with the following solution: CuSO₄·5H₂O, 0.5 gm; CrK(SO₄)₂·12H₂O, 0.5 gm; 10% acetic acid, 3 ml; and distilled water, 250 ml. Dehydration, clearing and covering complete the process. Myelin sheaths are stained bright blue; meninges and the adventitia of blood vessels are blue; red blood cells are green. Nissl material is stained brilliant red; axon hillocks, axis cylinders, ependyma, nuclei and some cytoplasm of neuroglia, media and endothelium of blood vessels are pink.     

Adaptation of the Millon, Sudan Black B, and Periodic Acid-Schiff Technics for Block Staining of Insect Tissues

Spermatophores and reproductive systems of the beetle, Lytta nuttalli Say, fixed in Bouin's aqueous picroformol or buffered 10% neutral formol were stained in toto by the Millon, Sudan black B and periodic acid-Schiff reactions as follows. Millon: after excess fixative is removed in 70% ethanol, specimens are brought to water, stained in Millon's reagent at 60 C for 1 hr, rinsed in 2% aqueous nitric acid at 40-50 C, dehydrated rapidly, cleared, embedded and sectioned as usual. Sudan black B: specimens are taken to absolute ethanol, stained in a saturated solution of Sudan black B in absolute ethanol at room temperature for 24-48 hr, rinsed and cleared in xylene, embedded and sectioned. PAS: specimens are brought to water, oxidized in 0.5 aqueous HIO₄ at 37 C for 30 min, washed in 2 changes of water, stained in Schiif reagent at room temperature for 1 hr, rinsed in 3 changes of 0.5% aqueous potassium metabisulfite, washed in running water for 10-15 min, dehydrated, cleared, embedded and sectioned. All 3 methods produced their characteristic staining in specimens up to 3 mm thick     

Different Patterns of Vein Loading of Exogenous [C]Sucrose in Leaves of Pisum sativum and Coleus blumei

Vein loading of exogenous [¹⁴C]sucrose was studied using short uptake and wash periods to distinguish between direct loading into veins and loading via mesophyll tissue. Mature leaf tissue of Pisum sativum L. cv Little Marvel, or Coleus blumei Benth. cv Candidum, was abraded and leaf discs were floated on [¹⁴C]sucrose solution for 1 or 2 minutes. Discs were then washed for 1 to 30 min either at room temperature or in the cold and were frozen, lyophilized, and autoradiographed. In P. sativum, veins were clearly labeled after 1 minute uptake and 1 minute wash periods. Autoradiographic images did not change appreciably with longer times of uptake or wash. Vein loading was inhibited by p-chloromercuribenzenesulfonic acid. These results indicate that uptake of exogenous sucrose occurs directly into the veins in this species. When C. blumei leaf discs were floated on [¹⁴C]sucrose for 2 minutes and washed in the cold, the mesophyll was labeled but little, if any, minor vein loading occurred. When discs were labeled for 2 minutes and washed at room temperature, label was transferred from the mesophyll to the veins within minutes. These results indicate that there may be different patterns of phloem loading of photosynthetically derived sucrose in these two species.     

T-system membranes in microsomal fractions of crayfish muscles: identification and differences in protein composition

Specific binding of 3H-ouabain and ruthenium red (RR) to membranes of T-tubules in crayfish muscles was used to identify the subfraction containing vesicles originating from the T-system. The microsomal fraction was prepared by differential centrifugation, and subfractions were separated in continuous or discontinuous sucrose density gradients. 3H-ouabain binding was estimated by scintillation counting; RR binding was examined by electron microscopy. The light subfraction was identified using both methods as that containing vesicles of T-tubules. Protein separation by SDS-electrophoresis revealed marked differences between the subfraction containing vesicles of T-tubules and other subfractions, the most distinctive feature being the presence of a protein of Mr 46,000 predominantly in the light subfraction.     

The adverse effects of HEPES, TES, and BES zwitterion buffers on the ultrastructure of cultured chick embryo epiphyseal chondrocytes

Chick embryo epiphyseal chondrocytes cultured in media containing HEPES, TES, and BES zwitterion buffers, used in combination or independently, consistently developed cytoplasmic vacuoles. This cytoplasmic vacuolation was resolved when the zwitterion buffered media was replaced by media containing bicarbonate:CO2 enriched air buffer. Vacuoles were infrequent or absent in cultures grown in bicarbonate:CO2 enriched air. Chondrocytes with an established extracellular matrix showed less vacuolation than fibroblastlike and polygonal shaped cells that lacked such a matrix. The granular endoplasmic reticulum and Golgi dictyosomes of zwitterion buffered chondrocytes were distended and contained a flocculent amorphous material. Cytoplasmic vacuoles (0.5 to 3.0 micron diam) formed by the fusion and intracellular accumulation of Golgi vesicles and vacuoles also contained a flocculent material enhanced by ruthenium red. Membrane bound extracellular vacuoles containing ruthenium red stained proteoglycan aggregates were common in the extracellular matrix of zwitterion buffered cultures but were generally absent from bicarbonate treated cultures. Electron dense calcium deposits seemed much larger and more numerous in the presence of zwitterion buffers. It is suggested that HEPES, TES, and BES buffers, used alone or in combination, may adversely affect cell membrane systems, and thus the transport or secretory mechanisms operative in cultured chondrocytes, or both, resulting in vacuole formation and the intracellular accumulation of synthesized export material. Although the mechanism by which HEPES, TES, and BES induce these changes remains unclear, the use of zwitterion buffers in biological preparations should be treated with caution.     

Rapid Chromosome and Sex-Chromatin Staining with Pinacyanol

This simple and reliable 10-min procedure for producing uniformly and intensely stained, as well as fade-resistant, chromosome and sex-chromatin preparations uses pinacyanol chloride as the dye. Slides are extracted in 5 N HC1 at 20-23 C for 2 min, washed in running tap water for 2 min, stained in 0.25% pinacyanol chloride solution (made up in 70% methanol) for 45 sec, differentiated in Wright's buffer solution (pH 6.4-6.5) for 45 sec, washed in running tap water for 5 sec, dehydrated in 2 changes, 1 min each, of absolute tertiary butanol, cleared in 3 changes of xylene, a minimum of 30 sec each, and mounted in a neutral synthetic resin such as Permount.     

Parathyroid cell variants may be provoked during immersion fixation

Summary Parathyroid glands of cattle, dogs, cats, mice and rats were immersed in glutaraldehyde or mixtures consisting of glutaraldehyde, formaldehyde and acrolein in either Na-phosphate, Na/K-phosphate or Na-cacodylate buffer, and postfixed with OsO₄ in the same buffers or, alternatively, in s-collidine.Excellent preservation of bovine, feline and murine parathyroid glands was achieved with fixation mixtures containing 1% glutaraldehyde, 1.5–2% formaldehyde and 2.5–5% acrolein in 0.1 M Na-cacodylate with or without Ca²⁺ and Mg²⁺, Na-phosphate or Na/K-phosphate at 4°C followed by postfixation with 1% OsO₄ in the same buffers or in s-collidine containing sucrose, Ca²⁺ and Mg²⁺. This procedure largely abolished the occurence of parathyroid cell variants. Bovine parathyroid glands were also satisfactorily preserved with 1% glutaraldehyde and 2% formaldehyde whereas 1% glutaraldehyde and 2.5 or 5% acrolein, lower or higher buffer osmolarity, or immersion at room temperature led to vacuolization of RER and to breakdown of membranes. In contrast, all fixation protocols led to the formation of dark and light cell variants and to multinucleated syncytial cells in dog and rat parathyroids. The results thus show that parathyroid cell variants arise during immersion fixation and that aldehydes, buffers and temperature are important factors for provoking parathyroid cell variants.     

Tissue fixation and osmium black formation with nonvolatile octavalent osmium compounds

Summary Several compounds of osmium^VIII, including potassium osmiamate and coordination complexes of OsO₄ with ammonia and various heterocyclic nitrogen compounds, have been synthesized and characterized. They have also been evaluated as substitutes for OsO₄ in postfixation of biological specimens and in light and electron microscopic cytochemical methods resulting in osmium black formation.The most useful of these osmic compounds, a molecular addition complex of hexamethylenetetramine (methenamine) with OsO₄, has a negligible vapor pressure of OsO₄. It has the molecular formula C₆H₁₂N₄.2OsO₄ and has been designated osmeth. Although it has only limited solubility, aqueous solutions of the compound (or of OsO₄) can be rapidly prepared by dissolution in a minimal amount of dimethylformamide and subsequent dilution with distilled water or buffer. Although stable in the solid state, the complex in solution undergoes partial dissociation releasing OsO₄, and the odor of OsO₄ becomes apparent.Such solutions of osmeth are (0.25%) considerably less concentrated with respect to OsO₄ than solutions (1–2%) ordinarily employed for ultrastructural preservation or in cytochemical studies. Osmeth has limited value for postosmication after glutaraldehyde fixation because the generation (release) of OsO₄ appears to be slow. Adequate osmication of tissue blocks exists only at the surface, but effective osmication can be achieved throughout tissue sections. In cytochemical reactions resulting in the formation of osmium blacks, the osmeth solutions are as effective as OsO₄ solutions of equivalent concentrations. Our findings indicate that OsO₄ solutions of less than 1% may be satisfactorily utilized in many cytochemical studies.Osmeth is safer and more convenient to handle than OsO₄ because small amounts may be solubilized as needed. It should be considered as a substitute for OsO₄ in ultrastructural cytochemistry.This investigation was supported by NIH research grant number DE 02668 from the National Institute of Dental Research and by NIH grant number RR 05333 from the Division of Research Facilities and ResourcesVisiting Professor, Dental Research Center, University of North Carolina at Chapel Hill, Jan.-May, 1975. Supported in part by USPHS Grant HD 09209     

RESULTS OF AN ATTEMPT TO OBTAIN PYRENOIDS OF ZYGNEMA BY BULK-ISOLATION METHODS1,2

Filaments of Zygnema were blended 3 min in a buffered sucrose solution, and a pyrenoid-rich cell fraction was obtained by filtration through a 10-μ filter. The filtrate was placed on a 35%/40%/50% (wt/wt) discontinuous sucrose density gradient and was spun at approximately 50,000 × g for 1/2–1 hr. Pyrenoids, which were collected at the 40%/50% interface, appeared with light microscopy to be firm, intact, spherical, colorless refractive bodies. Although in situ pyrenoids stained with propionocarmine, isolated pyrenoids would not stain, indicating a change in the chemical properties of pyrenoids during the isolation procedure.     

A calcium-binding sialoglycoprotein associated with an apparent eggshell membrane of Globodera rostochiensis

Energy dispersive X-ray microanalysis has been used to study the binding of two inhibitors of hatching in Globodera rostochiensis, ruthenium red and lanthanum, to a calcium binding site on the eggshell. Eggs were treated with either buffered ruthenium red or buffer only, and the levels of ruthenium and calcium that could be detected from the surface of eggshells were measured before and after repeated etching in a plasma oven. Counts for ruthenium increased during etching to a maximum after 8–12 min, just before the eggshell was eroded from the juvenile. The calcium levels on eggshells binding ruthenium red were suppressed relative to those from buffer. Similar experiments with lanthanum showed a significant increase in this inhibitor after 10 min etching and an accompanying suppression of calcium levels. The nature of the binding site was probed by measuring ruthenium red binding after pretreatment with selected enzymes. A non-specific protease, pronase, reduced the subsequent binding of this inhibitor but the site was resistant to trypsin in the presence of calcium. Neuraminidase also had an effect suggesting that the protein is a sialoglycoprotein. Phospholipase A2 but not Phospholipase C influenced the subsequent binding of ruthenium red. Phospholipase A₂ caused a loss of hatching ability and the degradation of a phospholipid from the eggshell. Ruthenium red binding was also reduced by treatment of eggshells with detergents or sonication. Apparently a calcium-binding sialoglycoprotein forms part of the integral proteins of an eggshell membrane located on the inner surface of the eggshell.