Ultrastructure and Capsule of Mycoplasma meleagridis

The ultrastructural study of Mycoplasma meleagridis utilized the electron microscope techniques of sectioning, histochemical staining, critical-point drying, and freeze etching. The predominant morphotype was a spherical form ranging in diameter from 200 to 700 nm. The other morphotypes were dumbbell-shaped cells interconnected by membranous tubules, and chains of streptococcal-like cells. These forms suggest replication by binary fission. An extracellular structure in the form of a capsular matrix was observed by staining with ruthenium red and potassium tellurite, and was also seen in specimens prepared by critical-point drying and freeze etching.     

Fractionation of the nucleus by divalent cations. Isolation of nuclear membranes

0.3–0.5 M MgCl₂ was used to disassemble nuclei and to isolate by a single centrifugation in less than 3 hr a nuclear envelope fraction in 55–60% yield as assessed by phospholipid recovery. Its gross chemical composition was determined and its morphology was studied electron microscopically by sectioning, freeze etching, and negative staining procedures.     

Surface structure of Uukuniemi virus.

Uukuniemi virus, grown in chicken embryo fibroblasts, has been studied by electron microscopy using negative staining, thin sectioning, and freeze-etching techniques. The spherical virus particle measures about 95 nm in diameter. Its envelope consists of a 5-nm thick membrane covered by 8- to 10-nm long surface projections. These are composed of two polypeptides species of about the same size. Both of them can be removed by digestion with the proteolytic enzyme thermolysin except for a small fragment. The enzyme-treated particles are smooth surfaced and extremely deformable. The glycopolypeptides are clustered to form hollow cylindrical morphological units, 10 to 12 nm in diameter, with a 5-nm central cavity. Both negative staining and freeze-etching suggest that these units are penton-hexon clusters arranged in a T = 12, P = 3, icosahedral surface lattice. The membrane to which the surface subunits are attached is probably a lipid bilayer as evidenced by its double-track appearance in thin sections and the tendency of the freeze fracturing to occur within it. The strand-like nucleoprotein appears from thin-sectioning results to be to a large part located in a zone underneath the membrane.     

Direct imaging electron microscopy (EM) methods in modern structural biology: Overview and comparison with X‐ray crystallography and single‐particle cryo‐EM reconstruction in the studies of large macromolecules

Determining the structure of macromolecules is important for understanding their function. The fine structure of large macromolecules is currently studied primarily by X‐ray crystallography and single‐particle cryo‐electron microscopy (EM) reconstruction. Before the development of these techniques, macromolecular structure was often examined by negative‐staining, rotary‐shadowing and freeze‐etching EM, which are categorised here as ‘direct imaging EM methods’. In this review, the results are summarised by each of the above techniques and compared with respect to four macromolecules: the ryanodine receptor, cadherin, rhodopsin and the ribosome–translocon complex (RTC). The results of structural analysis of the ryanodine receptor and cadherin are consistent between each technique. The results obtained for rhodopsin vary to some extent within each technique and between the different techniques. Finally, the results for RTC are inconsistent between direct imaging EM and other analytical techniques, especially with respect to the space within RTC, the reasons for which are discussed. Then, the role of direct imaging EM methods in modern structural biology is discussed. Direct imaging methods should support and verify the results obtained by other analytical methods capable of solving three‐dimensional molecular architecture, and they should still be used as a primary tool for studying macromolecule structure in vivo.     

Quantitative fluorescence spectrophotometry of acridine orange-stained unfixed cells. Potential for automated detection of human uterine cancer.

After staining with acridine orange (AO), the nuclei of unfixed cells from the human female genital tract exhibited the same fluorescence behavior previously observed for human and murine leukocytes and mouse ascites tumor cells. With staining conditions chosen to assure saturation of the green-fluorescing AO-nucleic acid complex in normal cells, corrected fluorescence emission spectra were recorded from the entire nucleus of 341 cells taken from 32 normal and 28 abnormal patients. Intensity of the recorded spectra was expressed in phosphor particle units, a fixed arbitrary unit of fluorescence intensity, to display intensity differences among the spectra from the various cell types. In all abnormal samples, one or more cells were found with 530-nm nuclear fluorescence intensity considerably greater than the maximum intensity recorded from normal cells. Determination of the adequacy of 530-nm nuclear fluorescence intensity as a criterion for cancer detection requires additional investigation. Additional criteria, if needed, may be supplied by the metachromasy of AO-stained unfixed cells.     

An alcohol-soluble Schiff's reagent: a histochemical application of the complex between Schiff's reagent and phosphotungstic acid.

A schedule for staining partially hydrated PAS-positive structures using non-aqueous solutions has been devised. Tissues are dewaxed, taken down to 70% alcohol, oxidised for 10 min in a 1% w/v alcoholic solution of periodic acid, treated with an alcoholic solution of phosphotungstic acid-Schiff reagent complex (PTA-Schiff reagent) for 25 min, washed in alcohol, cleared in xylene and mounted in a synthetic medium. The PTA-Schiff reagent complex prepared from de Tomasi Schiff reagent by precipitation with PTA may be stored in the deep freeze for many months and dissolved freshly in alcohol for use. The PTA-Schiff reagent used as above allows staining of highly water soluble materials such as dextran. From blocking and digestion studies the mode of action seems similar to de Tomasi Schiff reagent. The partial hydration of the tissues prior to reaction was found to be essential for effective staining.     

Cryofixation without cryoprotectants. Freeze substitution and freeze etching of an insect olfactory receptor

Antennae of the silk moth, Bombyx mori, were frozen by immersion into propane at ? 180 °C, and further processed by (a) freeze substitution (FS) or (b) freeze etching (FE). Although no cryoprotectant was used, freezing damage was observed in deeper tissue regions only. Data from FS specimens closely resemble those from FE replicas. Therefore, FS usually does not induce noticeable secondary artefacts by the preparation steps subsequent to freezing. When compared with chemically fixed antennae, the superior quality of cryofixation in this tissue is evident, particularly where cell surfaces and processes border the receptor lymph cavity: membranes are smooth following a steady course; dendrites and axons are round in cross-section with evenly distributed microtubules. The value of cryofixation is discussed with special reference to structures of presumed functional significance (e.g. stimulus conducting pore tubules, intramembrane particles of the receptor membrane, the ciliary segment of the dendrites, intercellular dilations, membrane junctions).     

A model mosaic membrane: Association of phospholipids and cytochrome oxidase

The structure and physical properties of model membranes formed from lipids and cytochromec oxidase have been examined. The lipid-depleted protein is in the form of 90 Å rods or globules. When phospholipid is added the rods swell and then. form sheets and concentric membrane vesicles. The protein is saturated with lipid at 65 g/atoms of phosphorus per mole of hemea. Electron microscope examination by negative staining, sectioning, and freeze etching indicates a 50 Å thick unit membrane with 50–60 Å protein globules in the lipid bilayer. Infrared, circular dichroism and fluorescence binding studies are consistent with globular protein units surrounded with lipid. Diolein will substitute for phospholipid but the membrane formed remains as sheets rather than vesicles. Saturated phospholipids will not interact with the oxidase to form membrane. The capacity to form membrane is specific to protein associated with the hemea, and other insoluble protein in the original oxidase preparation cannot form membrane.     

Effects of trehalose on stress tolerance and biocontrol efficacy of Cryptococcus laurentii

AIMS: To investigate the effects of internal trehalose on viability and biocontrol efficacy of antagonistic yeast Cryptococcus laurentii under stresses of low temperature (LT), controlled atmosphere (CA) and freeze drying. METHODS AND RESULTS: The content of trehalose in C. laurentii was increased by culturing the yeast in trehalose-containing medium. Compared with yeast cells with low trehalose level, the yeast cells with high level of internal trehalose not only obtained higher viability, but also showed higher population and better biocontrol efficacy against Penicillium expansum on apple fruit both at 1 degrees C and in CA condition (5% O(2), 5% CO(2), 1 degrees C). After freeze drying, survival of the yeast with high trehalose level was markedly increased when stored at 25 degrees C for 0, 15 and 30 days. Meanwhile, high integrity of plasma membrane was detected in the freeze-dried yeast with high trehalose level by propidium iodide staining. CONCLUSIONS: Induced accumulation of internal trehalose could improve viability and biocontrol efficacy of C. laurentii under stresses of LT and CA. Moreover, survival of the yeast was also increased as internal trehalose accumulation after freeze drying, and one of the reasons might be that trehalose gave an effective protection to plasma membrane. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this experiment show a promising way to improve the biocontrol performance of antagonistic yeasts under the commercial conditions.     

金鱼精子质膜和核膜的区域特异性

本文结合采用扫描和透射电子显微镜(包括冷冻断裂-蚀刻、超薄切片以及细胞化学染色法),研究了金鱼精子的超微结构特征,结果表明金鱼精子的质膜和核膜都具有区域特异性:1)精子质膜内大部分区域含有许多蛋白颗粒,但在特定区域内,蛋白颗粒呈有序排列,构成晶格状结构。2)精子头颈部和尾部均有液泡密集之处,凡是覆盖着液泡区的质膜内,几乎都不含有蛋白颗粒。3)液泡区能被细胞化学方法染成致密色,表明内含糖蛋白。4)核膜孔只集中存在于靠近颈部的核膜上,而其他部分则没有。本文对上述诸点进行了讨论。     

DNA Polymerase III holoenzyme of Escherichia coli. IV. The holoenzyme is an asymmetric dimer with twin active sites

Pol III, a subassembly of Escherichia coli DNA polymerase III holoenzyme lacking only the auxiliary beta subunit, was purified to homogeneity by an improved procedure. This assembly consists of nine different polypeptides, likely in a 1:1 stoichiometry: a catalytic core (pol III) of alpha (132 kDa), epsilon (27 kDa), and theta (10 kDa), and six auxiliary subunits: tau (71 kDa), gamma (52 kDa), delta (35 kDa), delta' (33 kDa), chi (15 kDa), and psi (12 kDa). The assembly behaves on gel filtration as a particle of about 800 kDa, indicating a content of two each of the subunits. A new procedure for purifying the core yielded a novel dimeric form which may provide the foundation for the dimeric nature of the more complex pol III and holoenzyme forms. Pol III readily dissociates into several subassemblies including pol III', likely a dimeric core with two tau subunits. The holoenzyme, purified by a similar procedure with ATP and Mg2+ present throughout, retained the beta subunit (37 kDa) as well as all the subunits present in pol III; the mass of the holoenzyme was estimated to be 900 kDa. The isolated initiation complex of holoenzyme with a primed template DNA and the elongation complex (formed in the presence of three deoxynucleoside triphosphates) had the same composition and stoichiometry as observed for pol III with two beta dimers in addition. An initiation complex assembled from a mixture of monomeric pol III core, gamma 2 delta delta' chi psi complex (gamma complex), beta, and tau retained the core, one beta dimer, and two tau subunits but was deficient in the gamma complex. When tau was omitted from the assembly mixture, the initiation complex contained one or two gamma complexes instead of the tau subunit. Based on these data, pol III holoenzyme is judged to be an asymmetric dimeric particle with twin pol III core active sites and two different sets of auxiliary units designed to achieve essentially concurrent replication of both leading and lagging strand templates.     

A time course study of water permeability and morphological alterations induced by mucosal hyperosmolarity in frog urinary bladder

Summary The role of the tight junction in the hydrosmotic response of the frog urinary bladder has been analysed by comparative kinetic studies and freeze etching examination. The comparison of the time course of the variations in transepithelial water net flux and of the alterations of tight junction ultrastructure in bladders exposed to mucosal hyperosmolar solutions shows that blisters are present in the tight junction before any increase in transepithelial water net flux. This indicates that the two phenomena are dissociated.In the same experimental conditions, freeze etching examination shows the presence in the tight junction of large areas of smooth and apparently stretched membrane where the typical network structure has disappeared. These alterations are reduced by further treatment with oxytocin and are probably not involved in the physiological hydrosomotic response.This work was supported in part by a grant from the Medical Research Council of Canada (J.H.).     

An alcohol-soluble Schiff's reagent: A histochemical application of the complex between Schiff's reagent and phosphotungstic acid

Summary A schedule for staining partially hydrated PAS-positive structures using non-aqueous solutions has been devised. Tissues are dewaxed, taken down to 70% alcohol, oxidised for 10 min in a 1% w/v alcoholic solution of periodic acid, treated with an alcoholic solution of phosphotungstic acid-Schiff reagent complex (PTA-Schiff reagent) for 25 min, washed in alcohol, cleared in xylene and mounted in a synthetic medium. The PTA-Schiff reagent complex prepared from de Tomasi Schiff reagent by precipitation with PTA may be stored in the deep freeze for many months and dissolved freshly in alcohol for use. The PTA-Schiff reagent sued as above allows staining of highly water soluble materials such as dextran. From blocking and digestion studies the mode of action seems similar to de Tomasi Schiff reagent. The partial hydration of the tissues prior to reaction was found to be essential for effective staining.     

State transitions and physicochemical aspects of cryoprotection and stabilization in freeze-drying of Lactobacillus rhamnosus GG (LGG)

Aims: The frozen and dehydrated state transitions of lactose and trehalose were determined and studied as factors affecting the stability of probiotic bacteria to understand physicochemical aspects of protection against freezing and dehydration of probiotic cultures. Methods and Results: Lactobacillus rhamnosus GG was frozen (–22 or –43°C), freeze‐dried and stored under controlled water vapour pressure (0%, 11%, 23% and 33% relative vapour pressure) conditions. Lactose, trehalose and their mixture (1 : 1) were used as protective media. These systems were confirmed to exhibit relatively similar state transition and water plasticization behaviour in freeze‐concentrated and dehydrated states as determined by differential scanning calorimetry. Ice formation and dehydrated materials were studied using cold‐stage microscopy and scanning electron microscopy. Trehalose and lactose–trehalose gave the most effective protection of cell viability as observed from colony forming units after freezing, dehydration and storage. Enhanced cell viability was observed when the freezing temperature was ?43°C. Conclusions: State transitions of protective media affect ice formation and cell viability in freeze‐drying and storage. Formation of a maximally freeze‐concentrated matrix with entrapped microbial cells is essential in freezing prior to freeze‐drying. Freeze‐drying must retain a solid amorphous state of protectant matrices. Freeze‐dried matrices contain cells entrapped in the protective matrices in the freezing process. The retention of viability during storage seems to be controlled by water plasticization of the protectant matrix and possibly interactions of water with the dehydrated cells. Highest cell viability was obtained in glassy protective media. Significance and Impact of the Study: This study shows that physicochemical properties of protective media affect the stability of dehydrated cultures. Trehalose and lactose may be used in combination, which is particularly important for the stabilization of probiotic bacteria in dairy systems.     

Ring formation of perfringolysin o as revealed by negative stain electron microscopy

Perfringolysin O revealed ring- and arc-shaped structures in the absence of cholesterol by negative staining electron microscopy, while before activation with cysteine it showed indistinct arcs and irregularly curved sticks but no rings. These structures were observed only at high concentrations (more than 17 000 hemolytic units per ml) and seemed to be particle associates with 20–28 particles (about 4 nm per particle) linked in a circle. The toxin produced an inactive and high molecular weight complex in the presence of phosphotungstic acid, which was isolated by Sephadex gel filtration. These findings suggest that the rings are the toxin-phosphotungstic acid complexes produced during specimen preparation on a grid in vacuo. The toxin lost the properties necessary for ring formation though moderate modification with glutaraldehyde, showing spindle- and egg-shaped particles of about 4 nm in minor and 5 nm in major axis by negative staining. These facts suggest that the aldehyde modifies the binding sites for phosphotungstic acid, which probably are the basic groups of the toxin molecules. In the presence of cholesterol, even at a low concentration, the toxin revealed rings and arcs by negative staining and also by carbon shadowing electron microscopy, although the toxin itself did not show any characteristic structure without phosphotungstic acid. These observations suggest that the rings are the toxin-cholesterol complexes themselves. The toxin-phosphotungstic acid complexes seemed to have a structure of a single layer of particle associates, while that of the toxin-cholesterol complexes may consist of double or triple layers of the associates because its border was thicker and more distinct.     

Quantitative immunogold localization of Na, K-ATPase along rat nephron.

Ultrastructural localization of Na, K-ATPase alpha-subunit along rat nephron segments was investigated quantitatively by immunogold electron microscopy on LR-White ultrathin sections using affinity-purified antibody against alpha-subunit of the enzyme. Ultrathin sections were incubated with the antibody at a saturation level and the number of gold particles bound per micron of the plasma membrane (particle density) of the tubular epithelial cells from the proximal tubule to the collecting duct was determined. In all the tubular epithelial cells, gold particles were located exclusively on the basolateral surface, and no significant binding of gold particles to the apical surface was observed. Distribution of gold particles on the basolateral membranes was quite heterogeneous; lateral membranes and infolded basal membranes were highly labeled, whereas the basal membranes which are in direct contact with the basal lamina were scarcely labeled. The average particle density on the basal surface was highest in the distal straight tubule cells (11.4 units), very high in the distal convoluted tubule cells (9.8 units), intermediate in the proximal tubule cells (3.3 units), in the connecting tubule cells (4.3 units), and in the principal cells of the collecting duct (5.6-3.8 units), low in the thin limb of Henle's loop (1.0 unit), and at the control level in the intercalated cells in the connecting and collecting duct. The relative number of gold particles/mm nephron segment and the relative number of gold particles in the various nephron segments were calculated using quantitative morphological data. The estimated distribution profile of the former was in good agreement with the Na, K-ATPase activity profile in rat nephron, which was determined biochemically with a microenzymatic method.     

New investigations on hematoxylin, hematein, and hematein-aluminium complexes. II. Hematein-aluminium complexes and hemalum staining.

The absorption spectra of hematein-aluminium solutions have been recorded at various concentrations and pH values; the solutions were prepared using analytically pure hematein and potassium alum as aluminium source. In aqueous solution, four different hematein-aluminium complexes could be distinguished by absorption spectroscopy. In weakly acidic media we observed the violet 1:1 and 1:2 complexes HmAl (VII) and HmAl2(3) (VIII), and in strongly acidic solution the red 1:1 complex HmAl2 (IX). Whereas, in weakly alkaline solution the blue 1:1 complex HmAl0 (X) was detected. By change of the pH value the complexes were mutual interconverted. The dye complexes were characterized by their absorption spectra and molar extinction coefficients. We have stained HeLa cells with the complex solutions under different experimental conditions. In all cases the nuclear staining was intense whereas the staining of the cytoplasm was weak. The microspectra of the stained nuclei were recorded and compared with the absorption spectra of the complexes in solution. Thus it was possible to identify the bound dye species. After staining in acidic media, the cells were red to red-violet depending on the reaction conditions. The three cationic dye species VII, VIII, and IX were bound in varying amounts. After blueing in weakly acidic media or in water, only the violet dye complex VII was detected whereas, after blueing in weakly alkaline media, only the blue complex X has been observed. Enzymatic digestion experiments have shown that the dye complexes in the nuclei were bound to DNA while those in the cytoplasm and nucleoli were bound to RNA. The binding between the dye complexes and the nucleic acids is discussed.     

Metabolism of Klebsiella pneumoniae freeze‐dried cultures for the design of BOD bioassays

Aims: The survival rate of freeze‐dried cultures is not enough information for technological applications of micro‐organisms. There could be serious metabolic/structural damage in the survivors, leading to a delay time that can jeopardize the design of a rapid biochemical oxygen demand (BOD) metabolic‐based bioassay. Therefore, we will study the metabolic activity (as ferricyanide reduction activity) and the survival rate (as colony‐forming units, CFU) of different Klebsiella pneumoniae freeze‐dried cultures looking for stable metabolic conditions after 35 days of storage. Method and Results: Here, we tried several simple freeze‐drying processes of Kl. pneumoniae. Electrochemical measurements of ferrocyanide and survival rates obtained with the different freeze‐dried cultures were used to choose the best freeze‐drying process that leads to a rapid metabolic‐based bioassay. Conclusions: The use of milk plus monosodium glutamate was the best choice to obtain a Kl. pneumoniae freeze‐dried culture with metabolic stable conditions after storage at ?20°C without the need of vacuum storage and ready to use after 20 min of rehydration. We also demonstrate that the viability and the metabolic activity are not always directly correlated. Significance and Impact of the Study: This study shows that the use of this Kl. pneumoniae freeze‐dried culture is appropriate for the design of a rapid BOD bioassay.     

Corbular sarcoplasmic reticulum of rabbit cardiac muscle

The structure of corbular sarcoplasmic reticulum as part of the sarcoplasmic reticulum (SR) in perfusion-fixed rabbit cardiac muscle was studied by thin sections and freeze fracture. In thin sections, processes on the surface of corbular SR have all the anatomical features of junctional processes of junctional SR. By freeze fracture, the E face of corbular SR was particle poor and showed deep pits; the P face was particle rich. The demonstrated structural homology of corbular SR to all forms of junctional SR justifies its inclusion in that group.     

Effect of culture medium and cryoprotectants on the growth and survival of probiotic lactobacilli during freeze drying

Aims:  To investigate the effects of the medium and cryoprotective agents used on the growth and survival of Lactobacillus plantarum and Lactobacillus rhamnosus GG during freeze drying.
Methods and Results:  A complex medium was developed consisting primarily of glucose, yeast extract and vegetable-derived peptone. Trehalose, sucrose and sorbitol were examined for their ability to protect the cells during freeze drying. Using standardized amount of cells and the optimized freeze drying media, the effect of the growth medium on cell survival during freeze drying was investigated. The results showed that glucose and yeast extract were the most important growth factors, while sucrose offered better protection than trehalose and sorbitol during freeze drying. When the cells were grown under carbon limiting conditions, their survival during freeze drying was significantly decreased.
Conclusions:  A clear relationship was observed between cell growth and the ability of the cells to survive during the freeze drying process.
Significance and Impact of the Study:  The survival of probiotic strains during freeze drying was shown to be dependent on the cryoprotectant used and the growth medium.