Multiple mitochondrial alterations in a case of myopathy.

Mitochondrial alterations are the most common feature of human myopathies. A biopsy of quadriceps muscle from a 50-year-old woman exhibiting myopathic symptoms was examined by transmission electron microscopy. Biopsied fibers from quadriceps muscle displayed numerous subsarcolemmal mitochondria that contained crystalloids. Numbering 1-6 per organelle, these consisted of rows of punctuate densities measuring ∼0.34 nm; the parallel rows of these dots had a periodicity of ∼0.8 nm. The crystalloids were ensconced within cristae or in the outer compartment. Some mitochondria without crystalloids had circumferential cristae, leaving a membrane-free center that was filled with a farinaceous material. Other scattered fibrocyte defects included disruption of the contractile apparatus or its sporadic replacement by a finely punctuate material in some myofibers. Intramitochondrial crystalloids, although morphologically striking, do not impair organelle physiology to a significant degree, so the muscle weakness of the patient must originate elsewhere.

High resolution scanning electron microscopy of the intracellular surface of intercalated disks in human heart.

The three-dimensional architecture of human cardiac intercalated disks was examined by high resolution scanning electron microscopy of osmium-macerated specimens. This methodology permits viewing of in situ intercalated disks from a vantage point inside individual cardiomyocytes. The erose nature of these structures was rendered in stark relief. Areas covered with clusters of particles were present on some membranous projections--these may represent a combination of desmosomes and fasciae adherentes. On the other hand, areas devoid of particles may correspond to gap junctions.

Gene expression, cell localization, and evolution of rodent submandibular gland androgen-binding protein.

A small dimeric androgen-binding protein (Abp) secreted by mouse (Mus) submandibular salivary glands has been hypothesized to function in mate-selection. The alpha-subunit (Abpa) evolves rapidly under natural selection. However, cellular site(s) of synthesis, mode of function, and patterns of evolution of this biologically important protein are otherwise unknown. We used a radiolabeled riboprobe and in situ hybridization to mouse (Mus) Abpa mRNA to localize Abpa synthesis to submandibular gland acinar cells. We next used a quantitative fluorescent thermal cycler (real-time PCR) to determine relative expression levels (normalized with a constitutively expressed ribosomal gene [S15/rig; rat insulinoma gene]) in male, female, and sexually immature European striped field mice (Apodemus agrarius). We hypothesized that gender or age-related differences might occur in production of a salivary protein related to sexual selection, but found no significant differences within our sample. Finally, we also used reverse transcription-PCR of mRNAs isolated from submandibular salivary glands to determine the Abpa allele in the striped wood mouse and compare it to published information on the homologue in Mus musculus domesticus.

Secretion by striated ducts of mammalian major salivary glands: review from an ultrastructural, functional, and evolutionary perspective.

In addition to their role in electrolyte homeostasis, striated ducts (SDs) in the major salivary glands of many mammalian species engage in secretion of organic products. This phenomenon usually is manifested as the presence of small serous-like secretory granules in the apical cytoplasm of SD cells. The composition of these granules is largely unknown, except in the case of the cat and rat submandibular gland, where the granules have unequivocally been shown to contain kallikrein. In some species, the apical cytoplasm of SD cells contains variable numbers of vesicles, both spherical and elongated, that vary in appearance from 'empty' to moderately dense. In the rat parotid gland, lucent vesicles transport glycoproteins to the luminal surface where they are incorporated into the apical plasmalemma and the glycocalyx. There is a strong possibility that in various species some of these vesicles are involved in transcytosis of antibodies to the saliva from their source (plasma cells) in the surrounding connective tissue. In addition, vesicles may engage in transfer of growth factors from the saliva to the interstitium. In a few species, conventional SDs have been replaced by ducts that are wholly given over to secretion, i.e., they entirely lack basal striations; although such ducts occupy the histological position of conventional SDs, it is not clear whether they represent a new type of duct or merely are modifications of SDs. Broad-based comparisons of ultrastructural and other data about SDs offer some insight into evolutionary history of salivary glands and their role in the adaptive radiation of mammals. Evolutionary patterns emerged when we made interspecific comparisons across mammalian orders. Among the bats, there is a clear relationship between SD secretion and general categories of diet.

Rat liver mitochondrial contact sites and carnitine palmitoyltransferase-I.

In hepatic mitochondria, the outer membrane enzyme, carnitine palmitoyltransferase-I (CPT-I), appears to colocalize with contact sites. We have prepared contact sites that are essentially devoid of noncontact site membranes. The contact site fraction has a high specific activity for CPT-I and contains a protein at 88 kDa that is recognized by antibodies directed at two different peptide epitopes on CPT-I. Similarly long-chain acyl-CoA synthetase (LCAS) specific activity is high in this fraction; a protein at 79 kDa is recognized by an antibody against LCAS. Although activity of carnitine palmitoyltransferase-II (CPT-II) is present, it is not enriched in the contact site fraction, and a protein of 68 kDa weakly reacted with anti-CPT-II antibody. Likewise, carnitine-acylcarnitine translocase (CACT) protein is present, but at a somewhat reduced level. Using an analytical continuous sucrose gradient, we demonstrate that the activities of CPT-I and LCAS and their associated immunoreactive proteins are present in a constant amount throughout the contact site subfractions. The enzymatic activity of CPT-II and its associated immunoreactive protein, as well as immunoreactive CACT, is absent in the lighter density gradient subfractions and is present in the higher density subfractions only in trace amounts. This heterogeneity of the contact site fraction is due to unvarying amounts of outer membrane and increasing amounts of attached inner membrane with increasing density of the subfractions.

Mitochondrial dysfunction in cardiac disease: ischemia--reperfusion, aging, and heart failure.

Mitochondria contribute to cardiac dysfunction and myocyte injury via a loss of metabolic capacity and by the production and release of toxic products. This article discusses aspects of mitochondrial structure and metabolism that are pertinent to the role of mitochondria in cardiac disease. Generalized mechanisms of mitochondrial-derived myocyte injury are also discussed, as are the strengths and weaknesses of experimental models used to study the contribution of mitochondria to cardiac injury. Finally, the involvement of mitochondria in the pathogenesis of specific cardiac disease states (ischemia, reperfusion, aging, ischemic preconditioning, and cardiomyopathy) is addressed.

Biodegradable fibres spun from poly(lactide) generated by reactive extrusion.

Poly(lactide) (PLA) was spun both in a high speed spinning process with take-up velocities of 1000-5000 m min(-1) and in a spin drawing process at draw ratios of 4-6. The effect of the melt spinning conditions on the development of the structural hierarchy in the fibres and the relations to the textile physical properties were investigated. The PLA fibres were characterised with regard to the degree of crystallinity by DSC and WAXS, the orientation by WAXS and birefringence, and the stress-strain behaviour. The maximum physical break stress and the E-modulus observed in the spin drawn fibres were about 490 MPa and 6.3 GPa, respectively, at an elongation at break of 30%. The PLA was a copolymer of L-lactide (92 wt.%) and meso-lactide (8 wt.%) and was generated by reactive extrusion polymerisation. The PLA virgin pellets were analysed regarding their degradation during the spinning processes. Their thermal and rheological properties were determined by DSC and dynamic rheological measurements, respectively, to derive suitable parameters for the melt spinning processes.

Scanning electron microscopy of the interior of cells in Hürthle cell tumors.

Four cases of Hürthle cell tumor were examined by scanning electron microscopy after being macerated to remove all soluble components. By all morphological criteria, Hürthle cells are oncocytes with their usual augmented complement of mitochondria. The Hürthle cell mitochondria either are ovate with central stacks of cristae or elliptical or rod-like with cristae that often are finger-like. As in salivary gland oncocytes, the shelf-like cristae are anchored to the inner boundary membrane by tubular necks. In some Hürthle cells, all of the mitochondria exhibit reticulate cristae. A few mitochondria harbor a globular inclusion in their inner compartment. The Golgi apparatuses are relatively simple, consisting of imbricated saccules that are edged by small, bud-like structures. The rare lumina in the midst of clusters of Hürthle cells are lined by numerous microvilli. Thus, scanning electron microscopy of macerated Hürthle cell tumors has revealed a number of features, especially of their mitochondria, that have escaped detection by transmission electron microscopy.

Ultrastructure of submucosal glands in human anterior middle nasal turbinates.

The abundant glands situated in the lamina propria of the human anterior middle nasal turbinate were complex tubules that consist of serous, seromucous, and mucous cells, either singly or in combination. Serous granules were homogeneously dense, but could have a small lighter core. Seromucous granules had a dense rim and a large compartment of appreciably lighter density. Gradation between serous and seromucous granules made precise identification of these secretory cell types difficult. Mucous cells were of conventional morphology. The secretory tubules, which possessed a complement of myoepithelial cells, gradually transformed into ducts or the changeover was relatively sudden. The ductular portions of the tubules consisted either of tall prismatic cells or of shorter columnar cells, both of which lacked secretory granules, but had many mitochondria in their supranuclear cytoplasm. In many cases the ducts, for most of their length, consisted of secretory cells. These glands clearly participate in the elaboration of the glycoconjugate coat that serves to protect the nasal mucosa and keeps it from drying out.

Organic secretion by striated ducts.

In addition to their role in electrolyte homeostasis, striated ducts in the parotid and submandibular glands of many mammalian species engage in secretion of organic products. This secretion usually is in the form of serous-like granules that lack substructure. Usually, the granules are in the 2.0-2.5 mm range, but granules smaller than 0.1 mm and larger than 12 mm have been observed. In mustelids, striated duct cells contain rhomboidal crystalloids in their apical cytoplasm; in dogs and at least two bat species, the apical plasmalemma is festooned with perpendicularly-oriented rods. Rather than granules, the supranuclear cytoplasm of duct cells in a number of species contains spherical or oblong vesicles. These may convey glycoproteins to the luminal surface where they are incorporated into the glycocalyx or the plasma membrane. Certain vesicles appear to be involved in the uptake of foreign proteins introduced retrogradely into the main excretory duct or of altered proteins produced by acinar cells in streptozotocin-induced diabetes.

Three-dimensional structure of oncocyte mitochondria in human salivary glands: a scanning electron microscope study.

Oncocytes occurring in otherwise normal human salivary glands were examined by scanning electron microscopy after being subjected to a maceration technique that removes all soluble cytoplasmic components and leaves only membranes in place. The three-dimensional images resulting from this procedure confirm that the majority of oncocyte mitochondria have shelf-like cristae that are attached to the inner boundary membrane, often by means of tubular structures. In some mitochondria, the cristae are digitiform and, when transected, give rise to putative vesicles. The oncocytic mitochondria in human salivary glands, by and large, lack the structural irregularity that has been reported for these organelles occurring in oncocytes in other organs.

Ultrastructure of the parotid gland in two species of naked-backed bats.

Naked-backed bats of the genus Pteronotus (family Mormoopidae) occur in the Neotropics from Mexico through northern South America. These are relatively small-sized insectivorous species that frequently roost in caves. Eight specimens of naked-backed bats (Pteronotus parnellii) were live-trapped in Suriname and one in Cuba (P. quadridens). Their parotid glands were fixed in an aldehyde mixture designed for field work and postfixed in the laboratory with osmium tetroxide. Tissues were further prepared for electron microscopy by conventional means. The parotid glands of the two species of Pteronotus closely resemble each other except for the substructure of their serous secretory granules. Serous granules in P. parnellii are bizonal, with a moderately dense inner matrix and an outer, denser corona or crescent. The matrix is occupied by laminae, flakes, and filaments in random array. In contrast, serous granules in P. quadridens consist of a uniform matrix that contains dense, usually stacked toroids or tubules either in random array or packed in bundles. A parotid gland from one specimen of P. parnellii contained an endpiece that consisted of cells that contained giant (up to 9 pm in diameter) serous granules. Serous cells in both species contain aggregates of small, uniformly dense, rod-like, membrane-delimited organelles as well as occasional bundles of cytofilaments. The endpieces are separated from intercalated ducts by a ring of granulated cells that contain secretory granules that often have a bull's-eye configuration. Intercalated and striated ducts are typical in appearance, except that many of the cells in the latter contain small, dense secretory granules in their apical cytoplasm. The parotid glands in the two species of naked-baked bats differ slightly in terms of acinar secretory granule ultrastructure, but otherwise are fairly conservative. It is thought that the glands in these particular bats might represent the "basal" condition of the salivary glands of insectivorous bats and thus can serve as a reference point for making comparisons to the highly diversified (in terms of diet) phyllostomid bats.

Plasticity and patterns of evolution in mammalian salivary glands: comparative immunohistochemistry of lysozyme in bats.

Salivary gland plasticity was a significant adaptive feature in the mammalian radiation. This plasticity is reflected in remarkable and well-documented interspecific phenotypic variation in gland ultrastructure and in the chemical components of saliva. However, comparative data are still too sparse for determination of evolutionary trends that combine phenotypic patterns with evolutionary history and the actual secretory products. Although our theoretical approach assumes that natural selection has taken advantage of salivary gland plasticity in gene regulation, gland development, and secretory cell organelles and processes, it still is difficult to delineate the biological roles of secretory products in the context of ecological adaptation. In the present investigation we used immunohistochemical techniques and a polyclonal antiserum against lysozyme to compare the parotid and principal submandibular glands in a set of 12 species of microchiropteran bats. With this data set we used comparative methods and phylogenetic trees to develop the foundations for testable hypotheses about the molecular genetics and adaptive significance of lysozyme production in bats. By comparing immunohistochemical results with ultrastructure, lysozyme-like immunoreactivity was associated with serous secretory granules in parotid gland acinar calls, parotid gland intercalated duct cells, and submandibular gland demilune cells. Lysozyme production in submandibular gland demilune cells marks a point of evolutionary divergence between three families of insectivorous bats and four families composed of species with diverse diets (ranging from carnivory to nectarivory). In terms of diet, lysozyme-like immunoreactivity corresponded most strongly with feeding on hard-bodied insects, leading to the hypothesis that lysozyme serves as an important chitinase in bat saliva.

Ultrastructure of major intraglandular ducts in the parotid gland of the African mole-rat.

The parotid gland of a female African mole-rat, Tachyoryctes splendens, was examined by light and electron microscopy. A pure seromucous gland of conventional histology, its striated and excretory ducts contain prominent cytoplasmic inclusions that are lightly stained with toluidine blue. At the ultrastructural level, the inclusions are seen to consist of a light to moderately dense structureless matrix in which are suspended scattered, randomly oriented filaments that either are 4-7 nm or approx 14 nm thick. A few multivesicular bodies or putative lipid droplets are sparsely distributed in the inclusions. Even though the inclusions lack an encompassing membrane, all other formed cytoplasmic structures are excluded. These inclusions are not present in the ducts of the submandibular gland of the mole-rat. The inclusions may confer special properties on the parotid gland of this fossorial animal that permit it to cope with the exigencies of a subterranean existence.

The developmental role of type III and type IV cells in the rat submandibular gland.

In addition to seromucous cells and a few mucous cells (Type II cells), submandibular glands of 2-8 month old rats contain two additional cell types: type III and type IV. Type III cells contain serous-type secretory granules that sometimes have a complex substructure; type IV cells appear to be seromucous, but their granules clearly are different from those in conventional endpiece seromucous cells. Both type III and IV cells are involved in histogenesis of new endpieces in a process that differs markedly from that occurring in perinatal glands. In this process, intercalated ducts bud and give rise to immature endpieces that consist entirely of type III cells. These differentiate into type IV cells, which in turn differentiate into standard seromucous cells. Concurrently, the intercalated ducts become shorter as their most distal cells differentiate into granular duct cells. This type of developmental process begins approximately 2 months postnatally, when histogenesis of endpieces by means of terminal tubules has ended, and continues until 6 months, when its frequency sharply declines.

An unusual parotid gland in the tent-building bat, Uroderma bilobatum: possible correlation of interspecific ultrastructural differences with differences in salivary pH and buffering capacity.

The tent-building bat, Uroderma bilobatum, is a small, frugivorous phyllostomid bat with a broad neotropical distribution. Generally found in humid forest, this bat lives in small groups that create daytime "roosts" from large leaves of a variety of tropical plants. Fruit eating engenders a variety of ecological and physiological challenges for bats, some of which could require adaptive features in their salivary glands. The parotid salivary glands of Uroderma bilobatum were prepared for transmission electron microscopy by using methods that have become standard for field work. The parotid gland is extremely unusual in structure. Although the secretory endpieces still produce serous granules with a complex substructure, they are modified into quasi striated ducts. Their basal folds, which are extensive, occasionally harbor some vertically oriented mitochondria, imparting a resemblance to striated ducts. Other evidence for the endpiece origin of these parenchymal components is a well-developed system of intercellular canaliculi, structures that never occur in bona fide striated ducts. The long but sparse intercalated ducts consist of two types of cells, each of which elaborates a modest number of secretory granules of differing substructure. Striated ducts are of conventional morphology, except that a few dark cells shaped like wine glasses are present in their walls. The striated duct cells produce no secretory granules, but their apical cytoplasm may contain some small, empty vesicles. Capillaries lie in longitudinal grooves in the base of the duct cells, an arrangement that might enhance electrolyte exchange. Excretory ducts consist of simple cuboidal epithelium composed of cytologically unspecialized cells that sometimes includes a dark cell. It was concluded that salivary glands could have a major role in adapting species to acquire nutrients from marginal sources, such as tropical fruits, which have a low protein and sodium content. The unusual parotid acinar cells in Uroderma bilobatum are discussed in the context of salivary pH and buffering capacity. Comparisons are made with four other bat species, including an insectivorous species with a salivary pH > 8.0 and a very high buffering capacity, an intermediate species, and a fruit bat with acidic-stimulated saliva and very low buffering capability. Such interspecific comparisons provide a foundation for hypothesizing that ultrastructural features of the acinar cell basolateral membranes and intercellular canaliculi correlate with differences involving Na/H+ exchangers and release of HCO3- and, thus, are associated with the species differences that are important to diet and nutrient acquisition.

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.

Cytomegalovirus infection in the submandibular and parotid salivary glands of an African grass mouse (Arvicanthus dembeensis).

The submandibular and parotid glands of one of five specimens of African grass mice (Arvicanthus dembeensis) were found to be infected with cytomegaloviruses, producing a profound cytomegaly in certain cells at the juncture of secretory endpieces and intercalated ducts. These cytomegalic cells tended to have multiple nuclei, many of which contained a characteristic reticular inclusion. The viruses appeared to arise in association with the intranuclear inclusions, then passed through the nuclear envelope to the cytoplasm where they budded into Golgi saccules or into small vacuoles, presumably of Golgi origin. Fusion of small virus-carrying vacuoles led to the formation of large vacuoles containing a plethora of viruses. Viruses were liberated into gland lumina via fusion of the vacuoles with the luminal plasmalemma. Fusion of vacuoles with dehiscent ones resulted in a form of chain exocytosis. The development of cytomegaloviruses in salivary glands may differ in details in a species-specific manner.

Intercellular material in the basal and lateral folds of parotid serous cells in four species of bats.

BACKGROUND: Basal folds are slender plications at the basal surface of acinar cells in the salivary glands of many mammalian species. These largely organelle-free folds increase the surface area of the basal plasmalemma manyfold and are unquestionably involved in the translocation of organic and inorganic molecules and water into the acinar cells. METHODS: Specimens of salivary glands were obtained from over 230 species of live-trapped bats from major areas of the globe. Tissues for electron microscopy were fixed and processed by conventional means. RESULTS: A number of the bat species examined had dense material in the intercellular spaces between basal and lateral folds of serous cells in the parotid gland. This intercellular material was particularly prominent in three species of New World bats, viz., Pteronotus parnellii, P quadridens, and Phyllostomus latifolius, and in one species of Old World bats, Chalinolobus argentatus. This dense material, which has a farinaceous texture, appears not to pass through tight junctions, so it is excluded from the lumina of intercellular canaliculi and acini. The dense material originates in the acinar cells--it is carried to the membranes of the folds via coated vesicles, which empty their dense content by exocytosis into the intercellular space. Similar dense material is present in the intercellular spaces of the basal labyrinth of striated ducts in the two species of Pteronotus. The manner in which this material accumulates in the striated duct is unclear. CONCLUSIONS: Although the function of the intracellular dense material is undetermined, it appears to be placed strategically to influence molecular traffic into acinar cells or to modulate the paracellular pathway. From a comparative evolutionary perspective, we hypothesize that, in bats, the combination of basal folds and extracellular densities is associated with insectivory. Similar morphologies appear to be lacking in frugivorous or nectarivorous species.