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PubMed1973

The organic-inorganic relationship in calcified mitochondria.

Experimentally induced calcification within mitochondria has been studied electron rnicroscopically. Cells investigated comprise hepatic cells damaged by CCl(4) intoxication, myocardial cells damaged by prolonged dihydrotachysterol (DHT) administration, and cells from skeletal muscle (gastrocnemius) damaged by DHT sensibilization and local injury. Cells from a human bowel carcinoma were studied too. Two types of intramitochondrial inorganic inclusion have been found. The first consists of clusters of apatite-like, needle-shaped crystals (crystalline aggregates), the second of clusters of very fine granules (granular aggregates). The former have been found mainly in mitochondria in apparently normal myocardial and muscular cells, the latter in mitochondria of degenerated hepatic, neoplastic, and myocardial cells. Crystalline aggregates are closely related to the membranes of cristae at first, but they later spread to occupy the whole mitochondrial matrix. Granular aggregates are initially found in the mitochondrial matrix near, but perhaps not touching, cristae; by growing they come into close contact with cristal membranes. Both types of aggregate show intrinsic electron opacity, which disappears after formic acid decalcification. Only the crystalline aggregates give an electron diffraction pattern of crystallinity. Uranium and lead staining of decalcified sections shows that both types of aggregate are intimately connected with an organic substrate. The substrate of crystalline aggregates consists of very thin, elongated structures shaped like the inorganic crystals. The substrate of granular aggregates consists of amorphous material gathered in clusters, with the same roundish shape and intercristal position as the inorganic granules. Both types of substrate are stained by phosphotungstic acid at low pH and by silver nitrate-methenamine after periodic acid oxidation. These results show that the organic content of the substrates includes glycoproteins; they have been confirmed by the periodic acid-Schiff (PAS) method under the optical microscope. These findings have been discussed in relation to the recent discovery of organic Ca(2+)-binding sites in mitochondria and to the general problems of soft tissue calcification.

Animals↗
PubMed1969

Application of standard micro-anatomical staining methods to epoxy resin-embedded sections.

Staining of semi-thin sections of osmium-fixed epoxy resin (Epon or Araldite)-embedded tissues with haematoxylin and acid dyes as counterstains (eosin or phloxin) was successfully achieved after intense oxidation using hydrogen peroxide as the oxygen donor. Results are closely similar to those obtained with paraffin sections (haematoxylin and eosin): nuclei stain blue, cytoplasm and extracellular structures (eg, collagen, elastica, basement membranes) stain with varying shades of red. Preliminary studies show that similarly good results, comparable with standard paraffinembedded histological materials, can be obtained, using H(2)O(2) pretreatment, with Weigert's iron haematoxylin, Mallory's phosphotungstic acid haematoxylin, Alcian blue, PAS reaction, Sudan III, Sudan black, oil red, Gomori's aldehyde fuchsin, and Baker's acid haematein.

Animals↗
PubMed1996

Stabilizing role of the basement membrane and dermal fibers during newt limb regeneration.

BACKGROUND: Following amputation of a newt limb, tissues at the amputation site undergo histolysis to give rise to a growth bud, or blastema, but they also provide a base on which the regenerate is constructed. Studies suggest that dermal tissues may differentially resist histolysis. METHODS AND RESULTS: To examine stability of tissues at the amputation site, more than 80 preblastemal staged regenerating limbs were examined histologically. Initially, all soft tissues not attached to bone retracted and were covered by migrating epithelium. The dermis was seen to be stable during the first week postamputation. Muscle dedifferentiated and was heavily stained with anti-tenascin antibodies, but the intact overlying dermis was unstained. Fiber bundles, revealed by staining with phosphotungstic acid hematoxylin, isolated the dermis from dedifferentiating deeper tissues during the first week postamputation, but partially broke down during the second week. However, the basement membrane (BM) remained as the distalmost intact structure at the amputation site in all limbs examined. The BM was the foundation for new BM synthesis which preceded dermis synthesis in the base of the blastema during the second week, even while undifferentiated cells were accumulating centrally. CONCLUSIONS: We suggest that the dermis resists histolysis long enough for new BM to form in continuity with that of the stump. Dermis formation (dermogenesis) distal to the amputation plane begins early as in mammalian healing but is not completed until after blastema formation. Thus, factors that inhibit dermal closure appear to distinguish regenerating from non-regenerating appendages.

Amputation, Surgical↗
PubMed1999

Multicentric oncocytoma of the lung diagnosed by fine-needle aspiration.

A 50-yr-old man presented with dyspnea. On chest X-ray, multiple pulmonary nodules were observed. Fine-needle aspiration biopsy (FNAB) showed tridimensional aggregates of atypical round epithelial cells, containing numerous cytoplasmic granules. The tissue fragment confirmed the presence of an epithelial tumor composed of trabecular sheets of clear cells, with numerous cytoplasmic granules which stained with phosphotungstic acid hematoxylin (PTAH). Immunohistochemically, the tumor cells were positive for cytokeratins and antimitochondrial antigen, whereas chromogranin, synaptophysin, S-100 protein, and HMB-45 were negative. Clinical and tomographic studies ruled out any tumor mass elsewhere. The rarity of this lesion in the lung and the potential difficulties for its diagnosis prompted us to report the clinical, cytological, and immunohistochemical findings in this case.

Adenoma, Oxyphilic↗
PubMed1996

Ultrastructure of nuclear condensation and localization of DNA and proteins in spermatozoa of a dasyurid marsupial, Sminthopsis crassicaudata.

In the dasyurid marsupial, Sminthopsis crassicaudata, the mature spermatozoon has an inner homogeneous (C1) and a peripheral indented (C2) region. Using DNase-gold conjugates, and biotinylated genomic DNA probes, DNA was found to occur in both C1 and C2 regions. The morphogenesis of the spermatozoon nucleus was investigated using ultrastructural and cytochemical studies. Spermiogenesis was divided into 15 steps. By step 10, condensation of the C1 region was complete, and at the caudal extremity of the spermatid nucleus, the nuclear envelope enclosed an electron-lucent space. This space and the surrounding nuclear envelope became very enlarged at step 11. At this stage, a plate of approximately 70 nm in thickness was present along the caudal segment of the C1 region; this "nuclear mantle" did not bind DNase-gold conjugates but stained for lysine-rich proteins using alcoholic phosphotungstic acid. Chromatin condensation resumed at step 12 with the appearance of spherical chromatin structures peripheral to the C1 chromatin. These structures then partially coalesced and the indentations of the C2 region were observed. The expanded nuclear envelope at the caudal extremity persisted in caput epididymal spermatozoa. Spherical inclusions within it did not bind to DNase-gold conjugates but stained for lysine-rich proteins. As the sperm traveled down the epididymis, these inclusions amassed near the nuclear pores and were then removed from the nucleus. In addition, the nuclear mantle was found to have disappeared by the time the spermatozoa reached the corpus epididymidis.

Animals↗
PubMed1975

Periodic fibrillar material in intracellular vesicles and in electron-dense bodies in chondrocytes of rat costal and tracheal cartilage at various ages.

The occurrence of intracellular fibrillar material (frequently banded) has been studied in normal costal and tracheal chondrocytes of rats at various ages ranging from 1 to 90 days. The study methods have included digestion with collagenase, electron histochemical techniques and routine electron microscopy. Banded fibrillar material has been observed intracellularly in vesicles or in electron-dense bodies in perichondrial and subperichondrial chondrocytes from rats of all ages. These fibrils and extracellular collagen fibrils are partially and equally degradable by collagenase, they are positive after staining with phosphotungstic acid or with silver nitrate methenamine, and their lucency corresponds with that of collagen when they are stained only with lead citrate. They have not been observed in intracellular clefts. They, therefore, seem to be formed intracellularly and to be exocytosed subsequently. Large vesicles and electron-dense bodies seem to be derived from Golgi saccules. A mechanism whereby banded intracellular fibrils could be formed from tropocollagen molecules is postulated. The frequency of occurrence and the diameter of intracellular fibrils seems to increase with increasing age.

Age Factors↗
PubMed1978

Ultrastructure of Octodon degus spermatozoon with special reference to the acrosome.

The ultrastructure of spermatozoa from the cauda epididymidis and vas deferens of Octodon degus-a Chilean hystricomorph rodent-is presented. The head of spermatozoa measured 7.7 micrometer long by 5.9 micrometer wide and the tail was 41 micrometer long. The head was flattened dorso-ventrally and ovate in outline. The acrosome was the most distinctive feature of O. degus spermatozoa. In a frontal view of the head, the rim of the acrosome surrounding the nucleus had the shape of an inverted U. The acrosomal region covering the plane of the flattened head exhibited dome-shaped protrusions. Transverse or sagittal sections of acrosomal protrusions showed that the plasma membrane and outer acrosomal membrane were evaginated, while the inner acrosomal membrane followed the contour of the nucleus. The protrusions were not distributed at random and they were absent in the equatorial segment and in the rim of the acrosome. In frontal views, near the boundary between the acrosome and post-acrosomal region, fine rods about 170 nm long ran obliquely on the caudal part of the equatorial segment. Behind the same boundary, the post-acrosomal region showed a serrated border. Phosphotungstic acid treatment at pH 0.3 produced staining at the surface of the sperm as well as within a superficial layer of the marginal thickening of the acrosome and on the acrosomal protuberances.

Acrosome↗
PubMed1983

Ultrastructural changes associated with the mineralization of deer antler cartilage.

The maturation and mineralization of deer antler cartilage were investigated ultrastructurally by using enzymatic digestions and subsequent staining with ruthenium red (RR) or phosphotungstic acid (PTA). RR staining of matrix granules was observed in the immature prechondroblastic matrix and became more intense as the cartilage matured into a mineralized tissue. The granules got larger and more numerically dense in the mature matrix. There were matrix granules that coalesced around matrix vesicles or remnants of such in the mineralized zone. These granules were observed after demineralization, and they were RR and acidic PTA-positive (they were not susceptible to hyaluronidase nor trypsin digestion, however). It appears that the granules were modified such that the matrix vesicle formed a centralized nidus for mineralization. The growth of hydroxyapatite crystals along matrix granules (which in this zone may or may not represent proteoglycan monomers) may have caused the coalescence. Microfibrils associated with matrix granules probably represented the hyaluronic acid core of the large proteoglycan complexes because of their susceptibility to hyaluronidase digestion.

Animals↗
PubMed1983

Synaptic changes in the hypothalamus of the prepuberal female rat administered estrogen.

Subcutaneous administration of estradiol benzoate (EB) to prepuberal female rats can advance vaginal opening, phasic pituitary gland luteinizing hormone (LH) secretion, and ovulation, presumably through a neural mechanism. This study investigated whether these effects are associated with changes in synaptic profiles in the arcuate nucleus of the hypothalamus (ARC) and the preoptic area (POA). Twenty-five-day-old female rats were administered EB, EB followed by progesterone on day 27, or oil vehicle alone; or they received no treatment. Blood was collected by jugular venipuncture at 1600 hr, on day 27, and plasma was assayed for LH by radioimmunoassay. Rat brains were immediately perfused for electron microscopy, and the ARC and POA were dissected out. Tissue blocks from these areas were processed with phosphotungstic acid for selective staining of neural synapses. Serum LH was markedly elevated in the EB-treated rats compared with controls. In the treated groups, LH values in serum were above 1,000 ng/ml, whereas the control values were less than 50. This acute rise of serum LH was accompanied by an acute increase of synaptic volume percent, area density, and numerical density in the ARC of EB-treated rats. The numerical density of the control groups was approximately 800 million observed synapses per cubic millimeter, whereas in the EB-treated groups, there were approximately 1.8 billion synapses per cubic millimeter. We found no differences in synaptic profiles of the POA in EB-treated animals as compared to the controls. We conclude from this study that estrogens act through neural mechanisms to accelerate maturation of neuroendocrine processes that govern phasic pituitary gland LH release and that this maturation process entails synaptogenesis in the ARC.

Animals↗
PubMed1975

Substructure in rod photoreceptor membranes.

Frog retinae, fixed only in buffered glutaraldehyde, were embedded for sectioning in glutaraldehyde polymerized with urea. In suitably thin sections globular substructures were seen in negative contrast after ionic staining with uranyl acetate and lead citrate, or after staining with neutralized phosphotungstic acid. Efforts to extract at least some of the lipid from sections before ionic staining enhanced the visualization of the "globules". Exposure to KMnO4 solution, used as an oxidative section stain, also outlined globular substructure in negative contrast, but with the additional feature that positively stained surface "leaflets" associated with the aqueous compartment were well defined. Staining sections with OSO4 vapor resulted in positively stained membranes, but without any evident substructure. However, when sections which previously had been exposed to OSO4 vapor were secondarily stained with uranyl acetate and/or lead citrate, positively stained globular substructures then were revealed. The globular substructures always were centered in the hydrophobic core region of the disc membranes, and symmetrically spanned the full thickness of this layer. The diameter of individual particles approximated 50-55 A. Reasons are presented for the supposition that the evident globules incorporate at least hydrophobic components of rhodopsin molecules. Findings are discussed in relation to various models of disc membrane organization that have been proposed in recent years.

Animals↗
PubMed1980

Genesis, ultrastructure and cytochemical study of the cat eosinophil.

Eosinophils from cat bone marrow and peripheral blood were studied by electron microscopy and cytochemical procedures. The maturation of eosinophils and formation of typical granules were described. Contrary to the accepted opinion that the core of animal's eosinophilic specific granules have a crytal-like structure, our observations revealed that the core has a myelin-like cylindrical appearance, whose layered formation proceeds from the inside outwards. Electron microscopic observations revealed that localization of reaction product to potassium pyroantimonate and phosphotungstic acid and to acid phosphatase activity was similar to that of eosinophils of man and other animals. Antimonate deposits and acid phosphatase activity were detected between the layers of the myelin-like structure of the core. Eosinophil granules failed to yield a positive reaction for peroxidase activity. The secretory activity of the eosinophil is discussed.

Acid Phosphatase↗
PubMed1981

Sequential events in the formation of collagen secretion granules with special reference to the development of segment-long-spacing-like aggregates.

The formation and maturation of collagen secretion granules in periodontal ligament (PDL) fibroblasts of young male Balb-C mice were studied by electron microscopy and cytochemistry. The Golgi apparatus was composed of several dictyosomes, each consisting of a stack of five smooth-walled cisternae. Each cisterna was connected at both ends to a dilated saccule. The cisternae, with their associated pairs of saccules, underwent progressive maturational changes from the immature to mature face of each dictyosome. The most immature saccules (type 1) were large, spherical, and filled with loosely arranged short filamentous structures. These saccules were continuous with the first and second Golgi cisternae (counting from the immature side). Golgi saccules type 2 were ellipsoidal, associated with the third and fourth cisternae, and contained parallel, elongate filaments. The most mature saccules, type 3, were more rectangular and connected by a short fifth cisterna. They contained aggregated filamentous material in the form of eight segment-long-spacing (SLS)-like crystallites, one in the center and seven at the periphery, to form a basic secretory unit. As type 3 saccules matured, the interconnecting cisterna progressively shortened until the two saccules were nearly juxtaposed. At this time the shortened fifth cisterna split to give rise to two independent presecretory granules. By progressive condensation, presecretory granules matured into secretion granules that contained a densely packed SLS-like aggregate, within which individual crystallites were no longer discernable. Maturation of cisternae and saccules involved removal of membrane, apparently by the formation and detachment of coated vesicles. The staining reaction with silver methenamine and phosphotungstic acid increased over the procollagen as the saccules matured, indicating addition of carbohydrate moieties and possible crosslinkages. It is concluded that the formation and maturation of collagen secretion granules in PDL fibroblasts involves the packaging and further modification of eight SLS-like crystallites, which are secreted as a basic unit.

Animals↗
PubMed1975

Ontogeny of monoamine neurons in the locus coeruleus, raphe nuclei and substantia nigra of the rat. II. Synaptogenesis.

Synaptogenesis was studied in the monoamine (MA) cell groups locus coeruleus (LC), dorsal and medial raphe nuclei (RN) and substantia nigra, zona compacta (SN) between day 18 of gestation and postnatal day 60 using ethanolic phosphotungstic acid (E-PTA) to visualize synaptic profiles. Nuclear area, and cellular packing density (inversely proportional to area of neuropil) were also determined. As determined using the E-PTA method, synaptogenesis begins in the neuropil of the SN first, on or before 18 days of gestation, and in the LC and RN at 19 days. Synaptogenesis on MA cell perikarya is first observed in the SN, on or before 18 days, and in the LC and RN at 20 days. The onset of somatic synaptogenesis coincides with the beginning of nuclear growth and development of the neuropil (decrease in cellular packing density) in all MA cell areas, raising the possibility of common factors in the initiation of these processes. Nonsynaptic contacts precede the appearance of synaptic profiles both in the neuropil and on the somata of the MA cells of the LC, RN and SN, and may represent precursors of mature synapses or desmosome-like contacts. Somatosomatic nonsynaptic contacts occur only prenatally between adjacent MA neurons in the LC, RN and SN. Although some synaptogenesis occurs prenatally in these MA cell groups (indiciating that these parts of the MA circuitry may be functional before birth), most of this synaptogenesis occurs postnatally and continues into adulthood. Since such synaptogenesis does not begin until 2-4 days prior to birth, whereas these neurons and their processes exhibit MA fluorescence as early as 12-14 days of gestation, they apparently are capable of synthesizing transmitter and proliferating terminals before they themselves are innervated.

Animals↗
PubMed1976

Early formation of synapses in the molecular layer of the fetal rat cerebellum.

The initial phase of synaptogenesis in the molecular layer of the albino rat cerebellum has been studied quantitatively and qualitatively with the electron microscope. Using both aldehyde-osmium (AL-OS) and ethanolic phosphotungstic acid (E-PTA) preparations, synapses were observed as early as embryonic day 19 throughout the entire rostro-caudal extent of the vermal molecular layer. The undeveloped nature of the elements participating in the formation of these synapses precluded their identification, for at this stage of cerebellar development, the Purkinje cells have not developed characteristic dendrites and both the massive production of cells within the external granule layer and the migration of neuroblasts through the molecular layer are in their initial phases. Evidence of synapse formation at this early stage of molecular layer development indicates that the rudiments of neuronal systems are present before the bulk of the molecular layer has come into existence and before most of the constituent elements of the cerebellum have differentiated. From embryonic day 19 to embryonic day 22, the number of E-PTA synaptic contacts was always less than the number of AL-OS synapses. This decrepancy and the variations observed in the morphology of the presynaptic components of E-PTA stained synaptic contacts, indicate that E-PTA morphology, and particularly the development of presynaptic dense projections, represents a late stage of synapse formation.

Animals↗
PubMed1995

Morphology of the release site of inhibitory synapses on the soma and dendrite of an identified neuron.

Synapses are complex arrangements of pre- and postsynaptic differentiations involved in neural communication. A key element in this synaptic transmission is the presynaptic active zone where the release of neurotransmitter occurs. Active zones can be visualized and analyzed after staining with ethanolic phosphotungstic acid (EPTA) on semithin (0.5 micron) sections. This staining has been used in association with postembedding immunogold labeling for the neurotransmitters glycine or GABA, to investigate the organization of chemically defined inhibitory active zones, viewed in their full extent, on different regions of the goldfish Mauthner (M-) cell. With this approach, a marked variability in size and shape was observed for the release sites contacting the different parts of the postsynaptic neuron. In the axon cap and on the soma, glycinergic afferent terminals have small presynaptic grids (0.066 +/- 0.029 micron2, n = 30 and 0.076 +/- 0.037 micron2, n = 46, respectively). These grids are quite circular and they include 12 to 13 presynaptic dense projections (PDPs). The situation is different on the lateral dendrite, where glycinergic and GABAergic active zones display a greater variability in their surface areas (mean = 0.147 +/- 0.100 micron2, n = 115 and 0.139 +/- 0.080 micron2, n = 125, respectively), and their number of PDPs (mean = 19 +/- 9) per individual grid. Similarly, the shape of the release sites over the dendrite is more complex (annular, horseshoe-shaped) when compared to those on the soma. These differences of dendritic versus somatic release sites could represent a structural basis to maximize the shunting effect of glycinergic and GABAergic inhibitory junctions, i.e., close to excitatory inputs. We also observed that the proportion of endings containing 1 or more active zones also varies. More precisely, 96% and 82% of glycinergic terminals in the axon cap and on the soma, respectively, display only one active zone. On the dendrite, their proportion falls to 65.5% for both glycine- and GABA-containing boutons. The remaining inhibitory terminals contain 2 (30%) and 3 to 4 (4.5%) presynaptic grids. These results reveal a greater variability of morphology and organization of the inhibitory release sites at dendritic versus somatic locations. The functional significance of this observation for the synaptic transmission is discussed.

Animals↗
PubMed2004

Salivary duct carcinoma with neuroendocrine features: report of a case with cytological and immunohistochemical study.

We report a salivary duct carcinoma (SDC) of parotid gland in a 75-year-old male. Initially, it was studied by fine-needle aspiration, which disclosed features of malignancy consistent with a high-grade carcinoma. Histologically, the tumor showed typical features of SDC, predominantly with a solid and apocrine pattern. The aggressive behavior of this tumor was documented by facial palsy and the presence of 12 regional lymph node metastases. Immunohistochemical study showed positivity for cytokeratins (AE1/AE3), cytokeratin 7, GCDFP-15, C-erbB-2, Mib-1, topoisomerase II alpha, p53, and androgen receptors. Diffuse positivity with chromogranin-A, synaptophysin, and Grimelius stains was also observed, suggesting endocrine features. Phosphotungstic acid hematoxylin, antimitochondrial antigen, progesterone and estrogen receptors, cytokeratin 20, and S-100 stains were negative. To our knowledge, this is the first case reported of SDC exhibiting neuroendocrine differentiation.

Aged↗
PubMed2006

Adult extracardiac rhabdomyoma: light and immunohistochemical studies of two cases in the parapharyngeal space.

BACKGROUND: We present two cases of adult rhabdomyoma in the parapharyngeal space. They are rare benign tumors with a characteristic histologic appearance. METHODS: The tumors were studied by light and immunohistochemical analysis using stains characteristic of striated muscle fibers. RESULTS: Cross-striation was demonstrated by phosphotungstic acid hematoxylin (PTAH), muscle specific actin, desmin, and myoglobin while dystrophin was expressed in the cell membranes. Clonal origin was confirmed by expression of myosin heavy chain-fast only. Expression of myosin-neonatal and myogenin proved slight proliferation with incipient differentiation in an otherwise mature tumor. CONCLUSION: The head and neck area harbors 90% of adult rhabdomyomas and should be considered in a differential diagnosis in this region. Immunohistochemistry confirms that the tumors are almost totally mature neoplasms of clonal origin.

Aged↗