Quantitative analysis of the mechanism of negative staining with native collagen fibrils and polar tropomyosin paracrystals

The mechanism of formation of the negatively stained image in electron microscopy was infestigated with native collagen fibrils as a model. The negatively stained image was simulated from the primary structure by using the values of volume or bulkiness of each amino acid residue as a parameter for stain-excluding capacity. The pattern simulated from the bulkiness values gave an excellent fit with the negatively stained image. Since some contribution of positive staining components to negative staining has been suggested, positive staining with uranyl acetate was tested with various washing solutions of different pH. While acidic conditions did not produce any stained image, a positively stained image was easily obtained at alkaline pH. On the other hand, negatively stained images with stains of different charge character remained essentially the same as those obtained with acidic uranyl stains. It was concluded that the contribution of positive components to the negatively stained image is negligible under the conventional conditions for negative staining with uranyl acetate. In order to demonstrate the utility of the analytical method employing the values of "bulkiness," we studied the unknown molecular packing in the polar lead paracrystal of rabbit skeletal tropomyosin. Utilizing the primary sequence data for alpha-tropomyosin we successfully showed the polar paracrystal to be an array of molecules which are parallel and in register. Further, our analysis made it possible to deduce the position of a given residue in the negatively stained pattern of the polar paracrystal.     

Electron microscopy and computer image averaging of ice-embedded large ribosomal subunits from Escherichia coli

Electron micrographs of frozen-hydrated, large ribosomal subunits from Escherichia coli have been analyzed by computer image processing. Images of subunits in the so-called "crown" orientation were analyzed by correlation alignment procedures developed for negatively stained specimens. Averages of the aligned images showed both similarities and differences to averages determined for negatively stained specimens. The L1 ridge is more dense and stalk-like in frozen-hydrated as compared with negatively stained subunits, possibly because it is associated with ribosomal RNA. The results show that it should be feasible to determine the three-dimensional structure of the large ribosomal subunit from micrographs of individual, frozen-hydrated subunits that have been tilted in the electron microscope.     

Electron microscopic studies of lipopolysaccharides from phase I and phase II Coxiella burnetii

Lipopolysaccharides from phase I (LPSI) Coxiella burnetii Ohio and Nine Mile strains and from phase II (LPSII) Nine Mile stain were negatively and positively and examined with the electron microscope. The ultrastructure of LPSI and LPSII positively stained with uranyl formate or uranyl acetate was ribbon-like. When negatively stained with uranyl acetate, LPSI was ribbon-like but LPSII exhibited hexagonal lattice structures. However, LPSII stained negatively with sodium phosphotungstate and ammonium molybdate exhibited hexagonal lattice ultrastructures which were not identical to those observed when negatively stained with uranyl acetate. The hexagonal lattice structures formed in vitro were due to the interactions of LPSII and the staining reagents rather than to protein-LPS interactions. The differences in the ultrastructures of LPSI and LPSII are undoubtedly based on variations in their chemical composition.     

QFH banding and DNA distribution in the macrochromosomes of the chicken Gallus domesticus

Chromosomes of Gallus domesticus were stained with rivanolum-SO2 (fluorescence Feulgen reaction) and by Hoechst 33258. Fluorescence photography was performed on a 35 mm film KN-3(KN-3) "Svema". The negatives were analyzed with the microdensitometer. The Feulgen (Fr--) and the Hoechst 3358 (H--) densitometric profiles of chromosomes showed light and dark segments along the metaphase chromosomes. The amount of DNA, as determined by the fluorescence Feulgen reaction, is not constant along the chromosome arms. Consequently, the base composition is not the only factor influencing the fluorescence of Hoechst 33258 along the chromosomes. The comparative analysis of densitometric profiles of the Hoechst 33258 and rivanolum-SO2 stained chromosomes shows that the Iq telomere band consists of GC-rich DNA (about 70% GC). The variation of DNA contents along the metaphase chromosome arms can be realized at both chromoneme and/or subchromoneme levels.     

Occurrence of microbodies in the green algaBracteacoccus cinnabarinus grown heterotrophically

Summary A comparative study was made of the ultrastructure and abundance of microbodies in the green algaBracteacoccus cinnabarinus grown photoautotrophically and heterotrophically on a conventional culture medium containing sodium acetate, potassium acetate and glucose. Several changes were observed in the cells maintained under these conditions. Most noticeably, cells grown on acetate in both light and dark were packed with lipid bodies. Microbodies were found to be closely appressed to the lipid bodies in cells grown heterotrophically in the dark on sodium acetate and potassium acetate. The average number of microbody profiles per cell was in general threefold greater in cells grown on sodium acetate than those grown on potassium acetate. No microbodies were observed in cells maintained photoautotrophically on the three carbon sources or in cells maintained photoautotrophically on Bristol's inorganic medium alone. Cytochemical staining with 3,3-diaminobenzidine indicated the presence of catalase in the microbodies. The presence of microbodies suggests that the organelle may be performing functions similar to glyoxysomes in higher plants, namely the net conversion to succinate of acetyl CoA derived from lipid degredation. It is also apparent thatBracteacoccus can grow well as a heterotroph in the dark when acetate is included in the culture medium as a source of carbon.     

Negative staining studied by TEM and STEM mass measurements: Preliminary observations on collagen sheets negatively stained with uranyl acetate

Quantitative mass measurements by dark-field scanning transmission EM and conventional bright-field transmission EM have been used to determine the increase in mass brought about by negative staining. pN-collagen (which forms sheets of uniform thickness and known mass per unit area) was used as a test specimen; the negative stain was uranyl acetate (1%, pH 4.4). The mass increase corresponded to the addition of roughly 8 uranyl acetate molecules per nm² for lightly negatively stained specimens; for heavily stained specimens, it was 30 molecules or more. The appearance of the image was related to the mass increase. This preliminary study shows that mass measurements can provide a basis for the quantitative interpretation of images from negatively stained specimens.     

High resolution electron microscope autoradiography on negative stained specimens

Summary A new method for applying the electron microscope autoradiography to negatively stained specimens is presented.Free ribosomes and ribosome crystals, labeled with ³H-uridine, have been isolated from the rat liver and from the whole hypothermic chick embryo, respectively. The samples, fixed and negatively stained with uranyl acetate, have been treated for autoradiography with the Kodak NTE nuclear emulsion.This method allows to obtain autoradiograms characterized by a very high resolution power of well stained specimens.The perspectives of this new field of ultrastructural investigation are discussed.     

Isolation and characterization of keratin-like proteins from cultured cells with fibroblastic morphology

Intermediate filaments (IF) isolated from a variety of cultured cells, conventionally described as fibroblasts, are composed predominantely of proteins of molecular weights of 54,000 and/or 55,000. Less than 15% of the protein found in native IF preparations from these cells is composed of three to four polypeptides of molecular weights 60,000- 70,000. We have investigated some biochemical and immunological properties of these proteins isolated from BHK-21 and mouse 3T3 cells. They are capable of forming paracrystals that exhibit a light/dark banding pattern when negatively stained with uranyl acetate. The dark bands are composed of longitudinally aligned approximately 2-nm-diam filaments. The center-to-center spacing between either dark or light bands is 37-40 nm. These dimensions are consistent with the secondary structure of IF polypeptides and suggest that the dark bands represent lateral alignment of alpha-helical coiled-coil domains. Immunoblotting, secondary structure, as well as amino acid composition data indicate that the 60,000-70,000-mol-wt paracrystal polypeptides are similar to keratin. Thus, polypeptides with biochemical and immunological properties of epidermal keratin are present in cells normally considered to be fibroblasts.     

Sodium dodecyl sulfate-gel electrophoresis: staining of polypeptides using heavy metal salts

Water-soluble salts of several heavy metals were examined for their ability to stain polypeptides resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Brief gel exposure (5 min or less) to cobaltous acetate or chlorides of copper, nickel, and zinc produced negatively stained protein patterns that were qualitatively indistinguishable from those of parallel gels stained with Coomassie blue R-250. Protein patterns could be visualized less than 1 min after treatment of gels with zinc chloride; the threshold of detection was estimated at about 10-12 ng protein on standard-size slab gels. Test samples including human erythrocyte membranes, sialoglycoprotein (glycophorin) extracts, and commercial molecular weight protein standards were used to establish the scope of these stains. Protein patterns visualized by the heavy metal salts were compared and contrasted with profiles seen with three widely used silver stains. Proteins from gels treated with copper or zinc chloride could be easily recovered by simple diffusion; this makes feasible both analytical and preparative electrophoretic applications of the staining procedure. A mechanism is proposed to explain the observed protein staining by heavy metal salts.     

Scanning electron microscopy of the infundibulum, ampulla, and eggs of mice

The objective of the study reported here was to use scanning electron microscopy (SEM) to discover and describe the details of three-dimensional profiles and the natural (not surgically disturbed) topography/location of the infundibulum in the mouse. It will help new investigators to more quickly identify the infundibulum for successful transfer of microinjected eggs through a small opening into the oviduct/ampulla of pseudopregnant female mice for producing transgenic mice. Results of the study also illustrate the geographic orientation and natural topographic features of the ovary, infundibulum, ampulla, oviduct, and uterus. The presence of cilia on the surface of the crown foldings in the longitudinal section of the infundibular head stained with 1% toluidine blue provided direct evidence that evagination of the internal cilia of the infundibulum/oviduct results in formation of the infundibular crown. The new observation of the narrowing region of the infundibular head after surgical removal of the crown also suggests that formation of the infundibular crown may have resulted from the "evagination process" of internal cilia of the infundibulum/oviduct surface. The results also provide new evidence that the crown, terminal opening, and appearance of the left and the right infundibula of the same mouse differ.     

Extraction of osmium-containing lipids by section staining for TEM

Postfixation with osmium-ferrocyanide or OSO4 renders lipid droplets in rat liver and kidney homogeneously electron dense without additional section staining. In sections of the same block that have been single stained by uranyl acetate or lead citrate, lipid droplets show a more electron translucent center surrounded by a dense rim. In sections double stained with uranyl acetate and lead citrate, lipid droplets frequently appear as empty vacuoles, from which the electron dense content has been completely extracted.     

Brazilin-Toluidine Blue O and Hematoxylin-Darrow Red Methods for Brain and Spinal Cord

Tissue fixed in 10% formalin, formalin-95% ethanol 1:s CaCO₂ or phosphate buffer neutralized formalin, or methanol-chloroform 2:1, was dehydrated and embedded in paraffin or double-embedded by infiltration in 1% celloidin followed by a chloroform-paraffin sequence. Sections were attached to slides with either albumen or gelatine adhesive and processed throughout at room temperature of 24-26 C. For either method, mordanting 30-60 min in 1% iron alum was followed by a 10 min wash in 4 changes of distilled water. For brazilin-toluidne blue O, myelin was stained for 20-60 min, depending upon section thickness, in a self-differentiating solution consisting of: 0.15% Li₂CO₃ 75 ml; 6% brazilin in 95% ethanol, 25 ml; and NaIO₃ 75 mg. After a thorough washing, Nissl material was stained for 3-8 min in a solution consisting of: 0.1 M acetic acid, 90 ml; 0.1 M sodium acetate, 10 ml; and 1% toluidine blue 0, 2.5 ml. For hematoxylin-Darrow red, myelin was stained for 2-6 hr in a self-differentiating solution consisting of: 0.15% Li₂,CO₃ 95 ml; 10% hematoxylin in 95% ethanol, 5 ml; and NaIO₃ 25 mg. After a thorough washing, Nissl material was stained for 20 min or less in a solution consisting of: 0.1 M acetic acid, 90 ml; 0.1 M sodium acetate, 10 ml; Darrow red, 25 mg. This mixture was first boiled, cooled to room temperature and filtered. In both methods, washing, dehydration, clearing, and mounting completed the process. In the brazilin-toluidine blue technic, myelin sheaths were stained reddish purple; neuronal nuclei light blue with dark granules of chromatin; nucleoli dark blue; and cytoplasm blue with dark blue Nissl granules. In the hematoxylin-Darrow red procedure, myelin sheaths were blue-black; nuclei light red with dark granules of chromatin; nucleoli almost black; and cytoplasm red with bright red Nissl granules.     

Characteristic views of Escherichia coli RNA polymerase core enzyme in the scanning transmission electron microscope

Eight characteristic views of Escherichia coli RNA polymerase core enzyme are presented which were found using multivariate statistical image processing of about 1000 individual molecular images. The data set was obtained from negatively stained specimens with a scanning transmission electron microscope (STEM) operated in dark field mode. The molecular projections were found to a resolution of around 30 A, thus visualizing new structural details of the core enzyme. An approximate twofold axis seems to relate the beta to the beta' subunit as well as both alpha units to each other.     

Immunocytochemical localization of Δ3, Δ2-enoyl-CoA isomerase and NADPH-dependent-2,4-dienoyl-CoA reductase in rat kidney

Localization of ³, ²-enoyl-CoA isomerase (ECI) and NADPH-dependent-2,4-dienoyl-CoA reductase (DCR) in the rat kidney was investigated by immunocytochemical techniques. The kidneys were perfusion-fixed and embedded in Epon or LR White. For light microscopy, semi-thin sections of Epon-embedded materials were stained by the immunoenzyme technique after the epoxy resin was removed by treatment with sodium ethoxide. For electron microscopy, ultra-thin sections of LR White-embedded materials were stained by the protein A-gold technique. By light microscopy, the S1 segment of the proximal tubule was most heavily stained for ECI and DCR whilst S2 and S3 segments showed intermediate staining. A weak staining reaction was observed in the distal tubule and the medullary collecting tubule. In the cortical collecting tubule, heavily stained cells were present between weakly stained cells. By electron microscopy, gold particles showing the antigenic sites for ECI were confined mainly to the mitochondria, but few particles were observed in the peroxisomes. Gold labeling for DCR was localized both in the mitochondria and the peroxisomes. The labeling intensity of the peroxisomes was much higher than that of the mitochondria. The results suggest that metabolism of unsaturated fatty acids occurs mainly in the mitochondria and the peroxisomes of the proximal tubule in the kidney.     

Use of Gallocyanin as a Myelin Stain for Brain and Spinal Cord

Tissue fixed in 10% formalin, formol saline, CaCO₃ or phosphate buffer neutralized formalin, Baker's formol calcium, Cajal's formol ammonium bromide, formalin-95% ethanol 1:9, formalin-methanol 1:9, Lillie's methanol-chloroform or Salthouse's formol cetyltrimethylammonium bromide was dehydrated and embedded in paraffin. Sections were attached to slides with either albumen or gelatine adhesive and processed throughout at room temperature of 22-25 C. Mordanting 30-60 min in 1% iron alum was followed by a 10 min wash in 4 changes of distilled water. Myelin was stained in a gallocyanin self-differentiating solution for 1-2.5 hr; thick sections requiring the longer time. The staining solution (pH approximately 7.4) consisted of Na₂CO₃, 90 mg; distilled water, 100 ml; gallocyanin, 250 mg; and ethanol, 5 ml. The ethanol was added to this mixture last, and after the other ingredients had been boiled and then cooled to room temperature. After a staining and thorough washing, Nissl granules were stained for 5-10 min in a solution consisting of: 0.1 M acetic acid, 60 ml; 0.1 M sodium acetate, 40 ml; methyl green, 500 mg. Washing, dehydration, clearing and mounting completed the process. Myelin sheaths were stained dark violet; neuronal nuclei, light green with dark granules of chromatin; nucleoli of motor cells and erythrocytes, dark violet; cytoplasm, green with dark green Nissl granules. The simple and reliable method can be adapted easily for use with automatic tissue processors.     

Fibroblast adhesion to RGDS shows novel features compared with fibronectin

As previously shown by others, the fibroblast attachment and spreading activity of fibronectin is mimicked by a short peptide (RGDS or longer) from the cell binding domain. Normal rat kidney fibroblasts showed similar attachment kinetics on either peptide GRGDSC or bovine plasma fibronectin and binding to either substratum was inhibited by peptide alone. We now demonstrate, however, considerable differences in biological activity between peptide and fibronectin. In particular, cells developed novel adhesion structures on peptide-coated substrata. Interference reflection microscopy showed a predominance of small round dark grey/black patches of adherent membrane ("spots") with relatively few focal adhesions, which occurred only at the outermost cell margins in contrast to their distribution in cells spread on fibronectin. The spots were resistant to detergent extraction and stained less strongly or not at all for vinculin. Electron microscopy in vertical thin section showed that the ventral surface of the cell was characterized by "point-contacts", corresponding in size to the spot structures seen by interference reflection microscopy, and which were only occasionally associated with microfilaments. Cells also required a higher substratum loading of peptide than fibronectin to promote spreading and proceeded to spread less rapidly and to a lesser extent, developing very few and extremely fine actin cables.     

Reversed phase chromatography of isoaccepting tRNA''s from healthy and crown gall tissues from Nicotiana tabacum.

RPC 5 (Reversed Phase Chromatography) of aminoacyl-tRNA's from healthy and crown gall (induced by Agrobacterium tume-faciens strain B6) tobacco tissues were compared for eleven amino acids. For ten amino acids: alanine, arginine, glutamic acid, glycine, isoleucine, leucine, lysine, methionine, tyrosine, and valine, no qualitative or quantitative differences could be detected between aminoacyl-tRNA's from both sources. Phenylalanyl-tRNA's from crown gall tissues gave two peaks on RPC 5; the minor early eluting species (peak 1) was always absent in elution profiles of phenylalanyl-tRNA's from healthy tissues or from tobacco leaves. After the "Y" base was removed by pH 2.9 treatment, peak 2 of phenylalanine tRNA was shifted to the position of peak 1.     

Electrophoretic analyses of alcohol dehydrogenase, aldehyde dehydrogenase, aldehyde oxidase, sorbitol dehydrogenase and xanthine oxidase from mouse tissues.

1. Cellulose acetate zymograms of alcohol dehydrogenase (ADH), aldehyde dehydrogenase, sorbitol dehydrogenase, aldehyde oxidase, "phenazine" oxidase and xanthine oxidase extracted from tissues of inbred mice were examined. 2. ADH isozymes were differentially distributed in mouse tissues: A2--liver, kidney, adrenals and intestine; B2--all tissues examined; C2--stomach, adrenals, epididymis, ovary, uterus, lung. 3. Two NAD+-specific aldehyde dehydrogenase isozymes were observed in liver and kidney and differentially distributed in other tissues. Alcohol dehydrogenase, aldehyde oxidase, "phenazine" oxidase and xanthine oxidase were also stained when aldehyde dehydrogenase was being examined. 4. Two aldehyde oxidase isozymes exhibited highest activities in liver. 5. "Phenazine oxidase" was widely distributed in mouse tissues whereas xanthine oxidase exhibited highest activity in intestine and liver extracts. 6. Genetic variants for ADH-C2 established its identity with a second form of sorbitol dehydrogenase observed in stomach and other tissues. The major sorbitol dehydrogenase was found in high activity in liver, kidney, pancreas and male reproductive tissues.     

ELECTRON MICROSCOPE STUDY OF MITOCHONDRIAL 60S AND CYTOPLASMIC 80S RIBOSOMES FROM LOCUSTA MIGRATORIA

Purified mitochondrial ribosomes (60S) have been isolated from locust flight muscle. Purification could be achieved after lysis of mitochondria in 0.055 M MgCl₂. Mitochondrial 60S and cytoplasmic 80S ribosomes were investigated by electron microscopy in tissue sections, in sections of pellets of isolated ribosomes, and by negative staining of ribosomal suspensions. In negatively stained preparations, mitochondrial ribosomes show dimensions of ~270 x 210 x 215 Å; cytoplasmic ribosomes measure ~295 x 245 x 255 Å. From these values a volume ratio of mitochondrial to cytoplasmic ribosomes of 1: 1.5 was estimated. Despite their different sedimentation constants, mitochondrial ribosomes after negative staining show a morphology similar to that of cytoplasmic ribosomes. Both types of particles show bipartite profiles which are interpreted as "frontal views" and "lateral views." In contrast to measurements on negatively stained particles, the diameter of mitochondrial ribosomes in tissue sections is ~130 Å, while the diameter of cytoplasmic ribosomes is ~ 180–200 Å. These data suggest a volume ratio of mitochondrial to cytoplasmic ribosomes of 1:3. Subunits of mitochondrial ribosomes (40S and 25S) were obtained by incubation under dissociating conditions before fixation in glutaraldehyde. After negative staining, mitochondrial large (40S) subunits show rounded profiles with a shallow groove on a flattened side of the profile. Mitochondrial small subunits (25S) display elongated, triangular profiles.     

Diel changes in the CO2 exchange rates of reproductive organs of the tropical tree Durio zibethinus

The daily variations in the in situ CO₂ exchange of the reproductive organs of Durio zibethinus trees, growing in an experimental field at University Putra Malaysia (UPM), were examined at different growth stages. Reproductive organs emerged on the leafless portions of branches inside the crown. The photon flux densities (PFD) in the chambers used for the measurements were less than 100 mol m^–2 s^–1 and were 40% of the PFD outside of the crown. The daytime net respiration rate and the nighttime dark respiration rate were higher at the time of flower initiation and during the mixed stages, when flower buds, flowers, and fruit coexist, than at the flower bud stage. The net respiration rate was lower than the daytime dark respiration rate at given temperatures, especially at the flower bud and fruit stages. Conversely, the net respiration rate was similar to the daytime dark respiration rate at the mixed stage. Photosynthetic CO₂ refixation reduced the daily respiratory loss by 17, 5, 0.3, and 24% at the flower bud, flower initiation, mixed, and fruit stages, respectively.