Bernard Tandler

A study of fixation of early amphibian embryos for electron microscopy.

A comparative study of fixation techniques was conducted on cleavage stage amphibian embryos. Different combinations of primary fixatives, buffers and postfixation procedures were tested, as well as variations in the duration and temperature of fixation. Differences in preservation resulting from individual fixation mixtures and procedures were examined. A new procedure is described which provided a uniform, excellent preservation of early embryos. The initial fixative consisted of 3% glutaraldehyde, 2% formaldehyde, 1 % acrolein, and 2.5% dimethyl sulfoxide (DMSO), in an 0.1 M cacodylate buffer. Postfixation with buffered 2% osmium tetroxide in sucrose was carried out for an extended period of time, up to 24 hours, ensuring adequate membrane contrast throughout the embryo. Procedures for proper rinsing of fixatives, and for embedding of these embryos are also described. Results are also presented which indicate that the DMSO-trialdehyde combination followed by postosmication has application to other embryonic organisms in addition to amphibians.

Biochemical differences between subsarcolemmal and interfibrillar mitochondria from rat cardiac muscle: Effects of procedural manipulations

Differences in oxidative metabolism between subsarcolemmal and interfibrillar heart mitochondria were investigated. Interfibrillar mitochondria oxidized substrates donating reducing equivalents at Complex I (NADH-CoQ reductase), Complex II (succinate-CoQ reductase), and Complex III (CoQH2-cytochrome c reductase) more rapidly than did subsarcolemmal mitochondria. There was no difference in oxidation of substrates entering the electron transport chain at Complex IV (cytochrome c oxidase). Differences expressed in normal-ionic-strength medium at Complexes II and III but not I were eliminated in low-ionic-strength medium. The concentrations of cytochromes and activities of NADH and cytochrome c oxidase were virtually the same in the two populations. In permeabilized mitochondria, activities of succinate-duroquinone and TMPD plus ascorbate oxidase were significantly lower in the subsarcolemmal mitochondria. Differences in membrane permeability between the populations were suggested by the greater permeability of subsarcolemmal mitochondria to exogenous NADH. The influence of isolation buffers and preparative procedures on the two classes of mitochondria were also examined. Characteristic biochemical and morphological properties of the two populations were unchanged by exposing each to the preparative procedure used to isolate the alternate population; the oxidative performance of the two populations cannot be equalized by experimental manipulation.

Dynamic organization of mitochondria in human heart and in myocardial disease

Heart mitochondria, which, depending on their location within cardiomyofibers, are classified as either subsarcolemmal or interfibrillar, are the major sources of the high energy compound, adenosine triphosphate. Physiological differences between these two populations are reflected by differences in the morphology of their cristae, with those of subsarcolemmal mitochondria being mostly lamelliform, and those of interfibrillar mitochondria being mostly tubular. What determines the configuration of cristae, not only in cardiac mitochondria but in mitochondria in general, is unclear. The morphology of cardiac mitochondria, as well as their physiology, is responsive to the exigencies posed by a large variety of pathological situations. Giant cardiac mitochondria make an appearance in certain types of cardiomyopathy and as a result of dietary, pharmacological, and toxicological manipulation; such megamitochondria probably arise by a combination of fusion and true growth. Some of these enlarged organelles occasionally contain a membrane-bound deposit of beta-glycogen. Those giant mitochondria induced by experimental treatment usually can be restored to normal dimensions simply by supplying the missing nutrient or by deleting the noxious substance. In some conditions, such as endurance training and ischemia, the mitochondrial matrices become pale. Dense rods or plates are present in the outer compartment of mitochondria under certain conditions. Biochemical alterations in cardiac mitochondria appear to be important in heart failure. In aging, only interfibrillar mitochondria exhibit such changes, with the subsarcolemmal mitochondria unaffected. In certain heart afflictions, biochemical defects are not accompanied by obvious morphological transformations. Mitochondria clearly play a cardinal role in homeostasis of the heart.

Proteinases of Pseudomonas aeruginosa evoke mucin release by tracheal epithelium.

We have determined the potential of exoproducts from pathogenic bacteria to stimulate the release of high molecular weight mucins from goblet cells of airway epithelium in a rabbit tracheal explant system. Culture supernatants from proteolytic strains of Pseudomonas aeruginosa and Serratia marcescens, but not supernatants from a number of non-proteolytic strains, released mucins from goblet cells. Highly purified elastase and alkaline proteinase from P. aeruginosa stimulated goblet cell mucin release in a dose-dependent fashion. Lipopolysaccharide, exotoxin A, and alginate of P. aeruginosa did not possess mucin release properties. Proteolytic activity was required for mucin release by P. aeruginosa elastase, but such release in goblet cells was not mediated by cyclic AMP. Morphologic studies suggested rapid release of mucins from goblet cells was response to elastase by a process resembling apocrine secretion. Several nonbacterial proteinases mimicked the effect of Pseudomonas proteases. These studies provide support for the hypothesis that bacterial and other play a role in the pathogenesis of mucus hypersecretion in acute and chronic lung infections.

Studies on Giant Mitochondria

Mitochondria, which play a cardinal role in the economy of the cell, are excellent morphological indicators of the state of health of cells. One of the ways in which these organelles respond to metabolic injury is to increase in size, often to a point where they merit the appellation of giant mitochondria or megamitochondria. Although quite large, even gigantic, mitochondria may occur in certain normal cells,’ it is those organelles that have become greatly enlarged as a result of pathological processes in typical cells such as hepatocytes or cardiomyocytes that are of concern in this review. Giant mitochondria have been reported to occur in variety of human diseases and in liver cells of aged humans2-I6 (TABLE 1). Such mitochondria are usually observed retrospectively in fixed specimens and, because of their source, rarely are available for experimentation. Obviously, giant mitochondria that are experimentally induced in laboratory animals are indispensable to the study of altered mitochondria1 morphology and function. Giant mitochondria have been experimentally produced in rodents, principally in their hepatocytes (but in other animals and cell types as well), by nutritional m a n i p u l a t i ~ n ’ ~ ~ ~ ~ (TABLE 2), pharmacological agents2949 (TABLE 3), ethanol intoxication5a4 (TABLE 4), and Giant mitochondria in liver cells have one of three basic morphologies, depending on the means whereby they are induced. Hepatic megamitochondria occurring in mice fed a riboflavin-deficient diet may be up to 10 pm in diameter” (FIG. 1). They tend to be spherical and are very similar to, although considerably larger than, normal-sized mitochondria in the same cells. Within a unit cross-section area, the number and distribution of cristae are the same regardless of organelle size, although some megamitochondria may contain a stack of cristae. Intramitochondrial dense granules are scattered throughout the matrix of the giant mitochondria, which is of normal density (FIG. 2). I n contrast, giant hepatic mitochondria (greater than 10 pm in diameter) resulting from feeding mice or rats r~ialarnide;~ ethidium or the copper-chelating agent, c ~ p r i z o n e * ~ ~ ~ (FIG. 3), or from injection with triamcinolone,68 have short cristae that are restricted to the organelle periphery (FIG. 4); the matrix, which is greatly expanded, is of near normal density but lacks membranes. It should be noted that these seemingly diametrically opposed arrangements of cristae are not invariably related to a particular method of megamitochondrial induction. When galactoflavin, a reversible antagonist of riboflavin, is added to a riboflavin-deficient diet, the appearance of symptoms of ariboflavinosis is greatly accelerated, with giant mitochondria being observed in mice as early as 13 days after initiation of the diet, (TABLE 5).

The Malonyl-CoA-sensitive Form of Carnitine Palmitoyltransferase Is Not Localized Exclusively in the Outer Membrane of Rat Liver Mitochondria

The data used to support the idea that malonyl-coenzyme A (CoA)-sensitive carnitine palmitoyltransferase (CPT-I) is localized on the outer mitochondrial membrane are based on harsh techniques that disrupt mitochondrial physiology. We have turned to the use of the French press, which produces a shearing force that denudes mitochondria of their outer membrane without the physiologically disruptive effects characteristic of phosphate swelling. Our results indicate that the mitoplasts contain just 15–19% of the outer membrane marker enzyme activity while retaining 85% of the total CPT activity and 50% of both CPT-I, as well as long-chain acyl-CoA synthase activity, the latter two supposed outer membrane enzymes. These mitoplasts were shown by electron microscopy to have the configuration of mitochondria that merely have been divested of their outer membranes. Carnitine-dependent fatty acid oxidation was retained in the mitoplasts, showing that they were physiologically intact. Moreover, protein immunoblotting analysis showed that CPT-I, as well as the inner CPT-II, was localized in the mitoplast fraction. The outer membrane fraction, which consisted of membrane “ghosts,” contained most (50–60%) of marker enzyme activity, monoamine oxidase-B and porin proteins, but only about 27–29% CPT-I activity. Because CPT-I and long-chain acyl-CoA synthetase appear to be associated with both inner and outer membranes, we postulate that these enzymes reside in contact sites, which represent a melding of both limiting membranes.

Fine structure of oncocytes in human salivary glands.

Oncocytes, cells displaying marked cytoplasmic acidophilia and granularity, are present in the acini and ducts of normal human salivary glands. These cells apparently originate by a process of sequential transformation of normal epithelial cells. In the early forms, there is a great increase in the number of mitochondria, which are typical in morphology. In later oncocytes, these organelles undergo striking changes in form and size. These mitochondrial changes are accompanied by the gradual disappearance from the oncocyte of other cytoplasmic membrane systems and of plasmalemmar specializations. It is suggested that the structurally modified mitochondria are biochemically deficient. Onkocyten finden sich sowohl in den Acini wie in den Ausführungsgängen normaler Speicheldrüsen. Sie besitzen ein eosinophil granuliertes Cytoplasma und sind wahrscheinlich Abkömmlinge normaler Drüsenzellen, aus denen sie sich über eine Reihe von Zwischenstufen entwickeln. Die Frühformen zeigen eine starke Zunahme der Mitochondrien. In den späten Stadien erfahren die Mitochondrien charakteristische Veränderungen in bezug auf Form und Größe, begleitet von einem Schwund der cytoplasmatischen Membransysteme und weiterer plasmacellulärer Spezialorganellen. Die geschädigten Mitochondrien dürften auch in ihrer biochemischen Leistung beeinträchtigt sein.

Microtubular structures associated with the acrosome during spermiogenesis in the water-strider, Gerris remigis (Say).

The ultrastructural organization of the acrosome during the course of spermiogenesis in the water-strider, Gerris remigis, has been studied in the electron microscope. The acrosome, which in the mid-spermatid is spherical and about 6–8 μ in diameter, becomes extremely elongated, until in the spermatozoon it is an attenuated, tapering rod measuring more than 2.5 mm in length. During this change in form, a system of parallel tubules appears within the acrosome. These acrosomic tubules measure about 130 A in diameter, are present throughout the length of the acrosomal structure, and are probably responsible for its rigidity. A second system of microtubules, which have been preserved only with aldehyde fixatives, surrounds the acrosome. They are about 220 A in diameter, and extend from a point near the insertion of the flagellum to about the midpoint of the acrosome. These cytoplasmic microtubules may also participate in maintaining the rigidity of the acrosome, and may be involved in the transport of newly synthesized protein and polysaccharide.

Ultrastructure of the human submaxillary gland

SummaryMyoepithelial cells in the human submaxillary gland are stellate in form, with long, tapering processes. They are interposed between the base of the secretory cells and the basement membrane, and are bound to the secretory cells by desmosomes. Their cytoplasm contains numerous myofilaments measuring approximately 40 Å in diameter, which frequently aggregate to form structures similar to the dark bodies seen in smooth muscle cells. The myofilaments are anchored to the plasma membrane by attachment devices. The myoepithelial cells are often accompanied by cells which have a similar shape, but possess an extremely electron-lucent cytoplasm that contains almost no organelles or inclusions. Electron microscopical observations indicate that these cells, termed clear cells, are transformed directly into myoepithelium, since all morphological intergrades between the two cell types have been recognized.

Epon-Maraglas Embedment for Electron Microscopy

There can be little doubt that Epon (Luft 1961) is currently the most widely used embedding medium for electron microscopy. While for the most part this embedding material is reliable, it has the disquieting tendency to fail occasionally to polymerize properly for no apparent reason. to counter this problem, several investigators have proposed that epoxide-anhydride ratios be taken into account to arrive at the best balance of ingredients for the final embedding medium (Coulter 1967, Burke and Geiselman 1971, Chang 1973). in an alternative solution, Mollenhauer (1964) suggested that the Epon be mixed with Araldite, and indeed, this combination has achieved some popularity. Epon-Araldite mixtures, however, have a relatively high viscosity in the unpolymerized state; this may slow permeation of tissue specimens.