The use of tannic acid for the ultrastructural visualization of hyaluronic acid

Summary This study describes a method, which makes use of tannic acid (2%) as a component of a paraformaldehyde-glutaraldehyde based fixative, to reveal the presence and ultrastructure of glycosaminoglycans in the extracellular matrix. The ultrastructure of the extracellular matrix in the stage 24 chick embryo wing is examined after fixation by several procedures. After fixation in the absence of tannic acid, the intercellular spaces contain little extracellular matrix, except for occasional fibrils (collagen?). On the other hand, when tannic acid is included in the primary fixative, the intercellular spaces contain considerable amounts extracellular matrix which includes 3±0.5 nm filaments, ±30 nm granules, as well as putative collagen fibrils. The 3±0.5 nm diameter fibrils are not observed when the limbs had been injected in ovo with Streptomyces hyaluronidase (specific for hyaluronic acid) prior to fixation. Furthermore, the 3±0.5 nm fibrils resemble authentic hyaluronic acid that had been fixed by the same procedure in the presence of tannic acid. Limbs treated with tannic acid after osmication contained only small amounts of extracellular material, which was confined largely to cell surfaces. These results demonstrate that the use of tannic acid in the primary fixative can serve as a useful method for the ultrastructural visualization of several extracellular matrix materials, including hyaluronic acid.This study was supported by NIH grant HD 05505     

Trypanosoma cruzi: ultrastructural changes produced by an anti-trypanosomal factor from Pseudomonas fluorescens

Trypomastigotes and amastigotes of Trypanosoma cruzi exhibited distinct ultrastructural alterations when treated with an extracellular lytic substance (anti-trypanosomal factor) produced by Pseudomonas fluorescens. Marked swelling of the parasites and detachment of the plasma membrane from the subjacent cytoplasm were observed after 15 min of treatment. After 3 hr, the nucleus was extensively damaged, the kinetoplast was indistinguishable, the mitochondrion was markedly swollen, the cytoplasm was disrupted, and the plasma membrane showed extensive blebbing and focal loss of subpellicular microtubules. These changes were progressive, as shown by the occurrence of parasite ghosts after 10 hr. Amastigotes exhibited an extremely swollen mitochondrion with disrupted internal structure, widening of the perinuclear space, and blebbing of the external nuclear membrane. The kinetoplast, however, remained clearly discernible. The drugs used today in controlling Chagas' disease are toxic. Therefore, there is a need for new anti-trypanosomal agents such as the Pseudomonas fluorescens antibiotics. The observations described in this study indicate the potential chemotherapeutic usefulness of these compounds for this disease.     

ROOT CONTRACTION IN HYACINTH. I. EFFECTS OF IAA ON DIFFERENTIAL CELL EXPANSION

Contractile roots of Hyacinthus orientalis L. cv ‘Pink Pearl’ shorten as a result of growth of inner cortical cells which expand radially and contract longitudinally. Brief treatment with IAA (indole-3-acetic acid—0.5 and 1.0 mg/1) induces subapical swelling, root cap proliferation and decreased rates of elongation in potentially contractile roots. Growth resumes with removal of IAA from the culture medium and contraction subsequently occurs. The pattern of subsequent contraction is affected by prior IAA treatment; contraction occurs in the normal manner both acropetal and basipetal to the points of IAA-induced swelling, but does not occur in the swollen region itself. Microscopic examination of the swollen region reveals that cells of the middle and outer cortex are radially expanded and longitudinally shortened relative to middle and outer cortical cells of contracted and uncontracted portions of the same root and control roots. In contrast, inner cortical cells in swollen regions of IAA-treated roots show approximately 50% less radial expansion than inner cortical cells of control contracted roots. Middle and outer cortical cells in the swollen region of IAA-treated roots undergo radial expansion, while middle and outer cortical cells in adjacent contracting zones are compressed by radially expanding inner cortical cells. Average volumes of cortical cells in the IAA-induced swollen region increased approximately two-fold when contraction occurred in adjacent regions. These results suggest that in hyacinth roots, under certain circumstances, inner and outer cortical cells alike possess the ability for growth reorientation and expansion. However, during the usual course of contractile root development, cells of the outer cortex are restricted in this ability, through an as yet unknown mechanism, and are passively compressed by the radially expanding inner cortical cells.     

Utilization of trehalose during Dictyostelium discoideum spore germination

An analysis of metabolism by measurement of respiratory quotient values indicates that reduced substances, such as lipids and/or amino acids, are the primary respiratory substrates of dormant Dictyostelium discoideum spores. The spores appear to consume both reduced substances and carbohydrates during the swelling stage of germination. The respiration of emerged myxamoebae is again dominated by the consumption of reduced substances. The pool of trehalose remains largely intact during heat-induced activation and also during postactivation lag. The initiation of spore swelling is accompanied by a decrease in the trehalose pool; the majority of trehalose is consumed before late spore swelling. Upon placing heat-activated spores under restrictive environmental conditions, swelling and trehalose hydrolysis are both prevented. Release from these conditions results in rapid swelling and hydrolysis of trehalose. Trehalase, the enzyme responsible for trehalose breakdown, is present in dormant spores at basal levels. This preformed enzyme is responsible for the hydrolysis of trehalose even though there is a significant increase in trehalase activity with the emergence of myxamoebae. RNA and protein synthesis inhibitors do not prevent trehalose hydrolysis or spore swelling. It is concluded that oxidation of reduced substances occurs in dormant, activated, and swollen spores, as well as in emerged myxamoebae of D. discoideum. Carbohydrate utilization dominates over the oxidation of reduced substances only during the swelling stage of germination.     

The effect of fixative tonicity on the myosin filament lattice volume of frog muscle fixed following exposure to normal or hypertonic Ringer

Synopsis Frog sartorius muscles have been fixed sequentially with acrolein and osmium tetroxide dissolved in vehicles of various tonicities, and the myosin filament spacings and sarcomere lengths measured with the electron microscope. From these dimensions the myosin unit-cell volume has been calculated and compared with X-ray diffraction data to determine the effect of fixation. In muscles soaked in normal Ringer and afterwards fixed using normal Ringer as a vehicle for the fixation agents, the unitcell volume undergoes a 10.4% reduction during the preparative procedure. Muscles soaked in hypertonic Ringer undergo a similar reduction in volume during fixation, provided hypertonic Ringer is used as the vehicle; if they are fixed in normal Ringer, the lattice swells during fixation, even if the change to the normal tonicity vehicle occurs after acrolein fixation. If blocks suitable for embedding are cut from the muscles before, rather than after, osmium fixation, more complex changes in intracellular dimensions may occur, including artefactual swelling of the T-system. It is concluded that fixation of tissues exposed to modifications of normal physiological solutions should be performed using the same modified solutions as fixative vehicles.     

Evaluation of fixative composition,fixative storage,and fixation duration on the fine structure and volume of root-cell nucleoli

Summary— The effect of various combinations of three fixative compositions (glutaraldehyde buffered in veronal acetate, cacodylate, and piperazine-N, N'-bis[2-ethanesulfonic acid]—PIPES], two fixative storage times (fresh vs 6 weeks), and two fixation durations (3 h vs 9 days) on nucleolar fine structure and nucleolar volume in three root cell-types of oat seedlings (Avena sativa L, cv Seger) were evaluated. All fixatives show overall good preservation of fine structure. Nucleolar components are distinct and well delineated in cells fixed in solutions buffered with either cacodylate or veronal acetate; the components are more condensed when preserved in fixative buffered with PIPES. Nucleolar volume is greatest in cells fixed in the cacodylate fixative, and smallest in those preserved in the PIPES fixative. Among the treatments tested, the PIPES fixative evidently best maintains nucleolar volume. Distracting particulate deposits are abundant on nuclei and nucleoli in cells preserved in the veronal-acetate fixative. Contrary to common assumptions, aging of buffered fixative at room temperature for 6 weeks seems to affect neither the general quality of cellular preservation nor the pH of the fixatives, although nucleolar volume is reduced by such treatment. Long-period fixation (9 days) results in destruction of membrane integrity (mitochondria, plastids, ER), and shrinkage of organelles from the cytoplasm. Nucleolar volume is reduced with prolonged fixation.     

An analysis of the contribution of the preparatory technique to the appearance of condensed and orthodox conformations of liver mitochondria

The structure and volume of isolated mitochondria embedded for electron microscopy during different respiratory states were analyzed in thin sections. Three different embedding methods were compared; osmium tetroxide fixation/acetone dehydration, glutaraldehyde fixation/acetone dehydration, and glutaraldehyde fixation-osmium tetroxide postfixation/acetone dehydration. Analysis of fresh mitochondria, isolated in a sucrose medium, revealed the presence of a homogeneous population with respect to structure when any of the three methods were applied. After fixation with osmium alone, or in combination with glutaraldehyde, nearly 100% of the mitochondria were in a "condensed" conformation. Mitochondria fixed with glutaraldehyde alone resulted in a population of mitochondria that had large spaces separating the two membranes of the cristae which corresponds to the condensed conformation as observed after osmium fixation. Transfer of the mitochondria to the incubation medium led to the appearance of two classes of mitochondria with respect to size. One class had a volume close to that observed when suspended in sucrose, and another class was present that was 30-45% larger. In osmium fixed or in double-fixed preparations, these small and large classes corresponded to "condensed" and "orthodox" forms of mitochondria respectively. When glutaraldehyde was used alone as the fixative, the two size classes were also present. However, the mitochondria were homogeneous with respect to structure. In these mitochondria, the width of the space that separated the cristae membranes had become reduced when compared to mitochondria suspended in sucrose. The two size classes were also present in samples of mitochondria prepared during both states 3 and 4. State 4 conditions did not lead to any significant increase of the number of condensed mitochondria. In state 3 preparations, 65-70% of the population were condensed. The condensed and orthodox forms could be related to normal and swollen forms of mitochondria. Conditions that led to a swelling also led to an increase in the number of orthodox mitochondria in osmium-fixed material. The different appearance of the mitochondria is explained by the different conditions for fixation of the mitochondria that exist when nonswollen and swollen mitochondria are fixed. This difference is particularly crucial in the case of osmium tetroxide due to the unique way this fixative, among generally used fixatives, denatures proteins.     

The fine structure of isolated Acetabularia nuclei

Summary Acetabularia nuclei were isolated in solutions of sucrose and fixed, dehydrated and embedded for electron microscopy using a number of techniques. The dimensions of the nucleic changed during all the procedures used, but the addition of calcium to the fixative prevented swelling. The structure observed in isolated nuclei was in good agreement with that seen in nuclei in intact cells and amputated rhizoids. A layer of cytoplasm about 0.1 thick was found on the outside of even the cleanest nuclei — this layer probably gives the nucleus protection when it is isolated.     

THE MORPHOLOGY OF THE SWELLING PROCESS IN RAT LIVER MITOCHONDRIA

Through the use of combined spectrophotometric and electron microscope techniques, large amplitude swelling of rat liver mitochondria has been described as an ordered sequence of ultrastructural transitions. Prior to the actual swelling, mitochondria undergo two major conformational changes: condensed to twisted form and twisted to orthodox form. This sequence is independent of (a) the nature of swelling agents and (b) the time of onset of swelling. Agents that delay the onset of swelling act to increase the duration of the twisted conformation. Agents that prevent extensive swelling hold mitochondria in intermediate conformations. Gross swelling, immediately preceded by a decrease in electron opacity of the matrix, involves the rupture of the outer membrane and expansion of the inner compartment of the mitochondrion.     

Swellographic Study of Peptide Resin Swelling Behavior during Solid Phase Peptide Synthesis

A representative array of peptide resin swelling data obtained using swellographic monitoring technique was analyzed to determine general tendencies in swelling volume dynamics in the course of Boc- and Fmoc-SPPS. Efficiency of the approach based on analyzing swelling volume changes (ΔV) as a function of nominal molecular weight change (ΔM) of the pendant peptide chain was demonstrated. The conclusion was made that a linear relationship between swelling volume and weight changes of peptide resin is a normal basic trend in line with results of early model studies carried out by Merrifield and coworkers. Excellent linear ΔV vs. ΔM correlations (r > 0.97) throughout SPPS for peptide resins swollen in DMF appeared to be typical of >60% of analyzed peptides and also are invariably observed for incorporation of the first four C-terminal amino acids and Boc-deprotected peptide resins swollen in 50% TFA-DCM. Formal theoretical interpretation of the observed regularities and anomalies of swelling volume dynamics and related solvation changes was made in terms of solvation numbers defined as a number of solvent molecules associated with a specific molecular segment of peptide-resin.     

Regulation of mitochondrial matrix volume

Mitochondrial volume homeostasis is a housekeeping cellular function essential for maintaining the structural integrity of the organelle. Changes in mitochondrial volume have been associated with a wide range of important biological functions and pathologies. Mitochondrial matrix volume is controlled by osmotic balance between cytosol and mitochondria. Any dysbalance in the fluxes of the main intracellular ion, potassium, will thus affect the osmotic balance between cytosol and the matrix and promote the water movement between these two compartments. It has been hypothesized that activity of potassium efflux pathways exceeds the potassium influx in functioning mitochondria and that potassium concentration in matrix could be actually lower than in cytoplasm. This hypothesis provides a clear-cut explanation for the mitochondrial swelling observed after mitochondrial depolarization, mitochondrial calcium overload, or opening of permeability transition pore. It should also be noted that the rate of water flux into or out of the mitochondrion is determined not only by the osmotic gradient that acts as the driving force for water transport but also by the water permeability of the inner membrane. Recent data suggest that the mitochondrial inner membrane has also specific water channels, aquaporins, which facilitate water movement between cytoplasm and matrix. This review discusses different phases of mitochondrial swelling and summarizes the potential effects of mitochondrial swelling on cell function. potassium homeostasis; depolarization; mitochondrial swelling     

Oxidative properties of swollen rat liver mitochondria.

Rat liver mitochondria undergo extensive swelling when they are incubated in hypotonic sucrose medium containing 5 mm P_i. After 30 min of swelling at 25 °C, a three- to fourfold increase in volume has occurred, accompanied by gross disorganization of the matrix as observed by electron microscopy. Succinate-supported respiration was unchanged, but the respiration of NAD-linked substrates was reduced and there was a complete and irreversible loss of phosphorylation in both cases. β-Hydroxybutyrate-supported respiration was regained completely on addition of NAD to the swollen mitochondria. α-Ketoglutarate- and malate + pyruvate-supported respiration was only partially restored by the addition of NAD. This inhibition of respiration in swollen mitochondria may be due to a disorganization of a putative complex of Krebs cycle enzymes on the inner surface of the inner membrane.     

Microbody proliferation and segregation cycle in the single-microbody alga Cyanidioschyzon merolae

The proliferation cycle of the microbody was studied in the primitive red alga Cyanidioschyzon merolae, which contains one microbody per cell. Cells were synchronized with a dark/light cycle, and the morphology of the microbody and its interaction with other organelles were observed three-dimensionally by fluorescence microscopy, transmission electron microscopy, and computer-assisted three-dimensional reconstruction of serial thin sections. The microbody in interphase cells is a sphere of 0.3 μm in diameter without a core. In M-phase, the microbody passes through a series of irregular shapes, in the order rod, worm, branched, H-shaped and dumbbell, and symmetric fission occurs just before cytokinesis. The microbody duplicates its volume in M-phase and three-dimensional quantitative analysis revealed that its surface area increases before its volume does. The microbody touches the mitochondrion and the chloroplast throughout its proliferation cycle, except briefly in interphase cells, winding around the divisional plane of the mitochondrion at one phase. Immunocytochemical labeling of catalase as a marker of matrix proteins of the microbody revealed that the duplication of catalase occurs in tandem with the volume increase. While no specific apparatus was identified in the microbody divisional areas, we identified an electron-dense apparatus about 30–50 nm in diameter between the microbody and the mitochondrion that may play a role in segregating the daughter microbodies. These results are the first characterization to show the morphological changes of one microbody in a one-microbody alga without proliferation-inducing substrates, which have been used in many studies, and clearly show that two daughter microbodies arise by binary fission of the pre-existing microbody. Received: 11 November 1998 / Accepted: 22 December 1998     

A Mordanting Fixation for Intense Staining of Small Chromosomes

A combination iron-mordant fixative in which propionic acid is substituted for acetic acid has been found useful in preparing small plant chromosomes for carmine stained squashes. Propionic acid is better than acetic acid because it holds more iron in stable solution. The fixative is a 3:1 mixture of 95% alcohol and pure propionic acid which contains 400 mg. of Fe(OH)₃ per 100 ml. of propionic acid. The latter is previously prepared by dissolving the dry freshly prepared Fe(OH)₃ in it. To each 10 ml. vial of fixative is added a few drops of carmine stain. Standard aceto-carmine squashes of material fixed in this mixture show quick intense staining and are especially useful for differentiated chromosomes at mitotic prophase.     

The mechanism and pathway of pH induced swelling in cowpea chlorotic mottle virus

Normal mode analysis based on a simplified energy function was used to study the swelling process of the icosahedral virus, cowpea chlorotic mottle virus (CCMV). Native state virus particles (coat proteins) of this T=3 icosahedral virus have been shown to undergo a large conformational change to a swollen state when metal ions are removed or the pH is raised. A normal mode analysis based on the native state capsid showed one preferential direction, a breathing mode, that explains the majority of the structural rearrangement necessary to bring the native structure close to the swollen state. From the native form of CCMV, the structure can be displaced along the direction of a single breathing mode by different amounts to create several candidate swollen structures and a putative pathway for virus expansion. The R-factor between these predicted swollen capsid structures and experimental electron density from cryoelectron microscopy (cryo-EM) measurements is then calculated to indicate how well each structure satisfies the experimental measurements on the swollen capsid state. A decrease of the crystallographic R-factor value from approximately 72% to approximately 49% was observed for these simple incremental displacements along the breathing mode. The simultaneous displacement of the native structure along other relevant (symmetric, non-degenerate) modes produce a structure with an R-factor of 45%, which is further reduced to 43.9% after minimization: a value in good accord with models based on the EM data at 28 A resolution. Based on the incrementally expanded structures, a pathway for the swelling process has been proposed. Analysis of the intermediate structures along this pathway indicates a significant loss of interactions at the quasi-3-fold interfaces occurs in the initial stages of the swelling process and this serves as a trigger for the compact to swollen transition. Furthermore, the pH dependent swelling appears to be triggered by the titration of a single residue with an anomalous pK(a) value in the unswollen particle.     

Glutaraldehyde fixation of sodium transport in dog red blood cells

The large increase in passive Na flux that occurs when dog red blood cells are caused to shrink is amiloride sensitive and inhibited when Cl is replaced by nitrate or thiocyanate. Activation and deactivation of this transport pathway by manipulation of cell volume is reversible. Brief treatment of the cells with 0.01-0.03% glutaraldehyde can cause the shrinkage-activated transporter to become irreversibly activated or inactivated, depending on the volume of the cells at the time of glutaraldehyde exposure. Thus, if glutaraldehyde is applied when the cells are shrunken, the amiloride-sensitive Na transporter is activated and remains so regardless of subsequent alterations in cell volume. If the fixative is applied to swollen cells, no amount of subsequent shrinkage will turn on the Na pathway. In its fixed state, the activated transporter is fully amiloride sensitive, but it is no longer inhibited when Cl is replaced by thiocyanate. The action of glutaraldehyde thus allows one to dissect the response to cell shrinkage into two phases. Activation of the pathway is affected by anions and is not prevented by amiloride. Once activated and fixed, the anion requirement disappears. Amiloride inhibits movement of Na through the activated transporter. These experiments demonstrate how a chemical cross-linking agent may be used to study the functional properties of a regulable transport pathway.     

A thermodynamic model describing the nature of the crista junction: a structural motif in the mitochondrion

The use of electron tomography has allowed the three-dimensional membrane topography of the mitochondrion to be better understood. The most striking feature of this topology is the crista junction, a structure that may serve to divide functionally the inner membrane and intermembrane spaces. In situ these junctions seem to have a preferred size and shape independent of the source of the mitochondrion with few exceptions. When mitochondria are isolated and have a condensed matrix the crista junctions enlarge and become nondiscrete. Upon permeation of the inner membrane and subsequent swelling of the matrix space, the uniform circular nature of the crista junction reappears. We examine the distribution of shapes and sizes of crista junctions and suggest a thermodynamic model that explains the distribution based on current theories of bilayer membrane shapes. The theory of spontaneous curvature shows the circular junction to be a thermodynamically stable structure whose size and shape is influenced by the relative volume of the matrix. We conclude that the crista junction exists predominantly as a circular junction, with other shapes as exceptions made possible by specific characteristics of the lipid bilayer.     

Biochemical lesions of respiratory enzymes and configurational changes of mitochondria in vivo

Summary Chick embryo heart fragments in primary hanging-drop culture were treated with sodium fluoroacetate to induce inhibition of aconitate hydratase, a mitochondrial enzyme of the tricarboxylic acid cycle. The mitochondria were analyzed in the living myoblasts by phase-contrast time-lapse cinemicrography. The results were recorded in a 16 mm film. After 20–30 minutes contact of the cells with the inhibitor some mitochondria became thickened and swollen. The swelling was polymorphous, asynchronous and reversible; the same mitochondrion could swell and shrink many times. Some mitochondria seemed not to respond to fluoroacetate and remained rod-like. Mitochondria appeared the only cell components to be morphologically affected by fluoroacetate and the changes were specifically caused by the inhibitor. The type of mitochondrial swelling differed from the large-amplitude respiration-dependent swelling of the isolated mitochondria in vitro and from the configurational changes of isolated mitochondria associated with the respiratory states. The evidence pointed to a specific connection between the biochemical lesion caused by fluoroacetate and the configurational changes of the mitochondria. The mitochondrial swelling was to a large extent reversed by washing the cultures with Tyrode physiological saline solution and the reversal was further accentuated by incubation of the cultures in fresh nutrient medium.This work was supported by grants of the Consiglio Nazionale delle Richerce of Italy to both Institutes.     

The ultrastructural characters of the mature spermatozoon of Opechona bacillaris (Molin, 1859) (Digenea,Lepocreadiidae) a parasite of Scomber colias Gmelin, 1789 (Scombridae) off the coast of Dakar (Senegal)

This study presents the ultrastructure of the mature spermatozoon of Opechona bacillaris, a digenean belonging to the family Lepocreadiidae. The sperm cell of O. bacillaris exhibits the general pattern described in most of the Lepocreadioidea: two axonemes of the 9 + ‘1’ pattern of the Trepaxonemata, mitochondria, a cortical mitochondrion, a nucleus, electron‐dense material in the anterior extremity of the spermatozoon, external ornamentation of the plasma membrane with associated spinelike bodies, and granules of glycogen. However, particularities of O. bacillaris are the simultaneous presence in the anterior extremity of the spermatozoon of the electron‐dense material, a mitochondrion, and the absence of cortical microtubules. In the Lepocreadiidae, we describe for the first time in O. bacillaris spinelike bodies associated with the external ornamentation of the plasma membrane and two mitochondria. The first mitochondrion is moniliform and composed of a mitochondrial cord with joined mitochondrial bulges. The second mitochondrion shows a regular form. The posterior tip of the spermatozoon has only singlets to owing to the disorganization of the second axoneme and granules of glycogen as occurs in Hypocreadium caputvadum, the other studied species of the family Lepocreadiidae.