Osmiophilic Material Research Articles

Nitric oxide synthase-containing neurones and nerve fibres within cardiac ganglia of rat and guinea-pig: an electron-microscopic immunocytochemical study.

The nitric oxide synthase-immunoreactivity and NADPH-diaphorase activity of intracardiac neurones in the rat and guinea-pig was studied at the ultrastructural level. While some nitric oxide synthase-containing intracardiac neurones were very heavily labelled, with electron-dense immunoprecipitate distributed throughout the neuronal cell bodies and their processes, most of the labelled neurones exhibited a lighter and more patchy distribution of nitric oxide synthase-immunoreactive material. Synapses made by nitric oxide synthase-negative nerve fibres with labelled intracardiac neurones were seen. Conversely, many nitric oxide synthase-containing nerve fibres that made synaptic contacts with unlabelled intracardiac neurones were also observed. Some small granule-containing cells were nitric oxide synthase-immunoreactive and were associated with unlabelled nerve terminals, while non-immunoreactive small granule-containing cells that were innervated by nitric oxide synthase-immunoreactive nerves were also seen. Small patches of osmiophilic electron-dense material were observed in the cytoplasm of NADPH-diaphorase-positive intracardiac neurones. This is the first description of the ultrastructural distribution of nitric oxide synthase-immunoreactivity and NADPH-diaphorase activity in a subpopulation of intracardiac neurones of rat and guinea-pig heart and provides further evidence in support of a role for nitric oxide in the local control of the heart by intrinsic neurones.

Pathway of terpene excretion by the appendix of Sauromatum guttatum.

Electron microscopy of the cells of the thermogenic appendix of Sauromatum guttatum has revealed a fusion event between pocket-like structures of the rough endoplasmic reticulum (rER) and the plasma membrane. As a result of the fusion event, many regions of the plasma membrane have paired unit membranes (four leaflets instead of two). The fusion allows the transfer of osmiophilic material from the rER pockets to the plasma membrane, where the osmiophilic material is confined to bilayer, pocket-like structures. A clear correlation is found between the presence of the osmiophilic compound and sesquiterpenes. Prior to heat production, the rER- and plasma-membrane pockets are electron dense, and sesquiterpenes are detectable only in tissue extracts. On the day of heat production, electron-translucent pockets are subsequently found and the stored sesquiterpenes are released to the atmosphere. Three sesquiterpenes have been identified by gas chromatography-mass spectrometry as alpha-copaene and beta- and alpha-caryophyllene.

How Does Armeria maritima Tolerate High Heavy Metal Concentrations?

The perennial plant Armeria maritima ssp. halleri , growing on the copper-rich soil of a medieval mine dump, tolerates high heavy metal (HM) concentrations and accumulates e.g. 2000 (leaves) to 4000 (roots) times more copper in comparison to plants growing under normal conditions. For copper, as an example, the mechanisms of the HM-tolerance are discussed. The HM-tolerance of Armeria is the result of morphological differentiations and biochemical alterations. As measured by EDX analysis a great part of the copper in roots and leaves is retained in vacuoles of idioblasts («tannin cells») containing homogeneous or flaky precipitates of osmiophilic material. In the homogeneous precipitates, high copper concentrations can be measured. EELS spectra revealed that the copper in these vacuoles is chelated by polyhydroxy phenolic compounds. Moreover, a complex mixture of phenolic compounds from leaves and roots of Armeria can be separated by HPLC. Copper ions reaching the vascular bundle are translocated via the transpiration stream into the leaves and are excreted partly by salt glands on both leaf surfaces. Crystals on the surface of the leaves contain besides P, S, Cl, K and Ca large amounts of Cu and to a smaller extent Zn, Ni, Fe and Mn. A significant part of the copper in roots and leaves is found to be localized in cell walls, in the cytoplasm, in the stroma of the plastids and in the chromatin of the nuclei. In these compartments the copper is bound preferentially to proteins, as determined by EELS. Obviously, the copper in the cytoplasm represents a stress situation for the cell, resulting in an expression of heat stress proteins (HSP).

Endothelial cells injured by oxidized low density lipoprotein

Cultured endothelial cells from bovine aorta were exposed to oxidized low density lipoprotein and examined by electron microscopy. The endothelial cells contracted slightly and the intercellular junctions became unclear. Some osmiophilic material increased in the cytoplasm. The oxidized low density lipoprotein appears to injure endothelial cells and thereby plays a causative role in atherogenesis and thrombogenesis.

Iatrogenic Compressive Lumbar Myelopathy and Radiculopathy in Adult Cattle following Injection of an Adjuvanted Bacterin into Loin Muscle: Histopathology and Ultrastructure

Compressive lumbar myelopathy is a recognized iatrogenic complication of injecting water-in-oil vaccines into paravertebral sites of laboratory animals and chickens. Herein, we report the histologic and ultrastructural features of a similar complication in a herd of cattle. Iatrogenic posterior paresis developed over 34 days in 56 of 610 cows (9.2%) following injection of a commercial bacterin 11-34 days earlier into M. longissimus lumborum. The bacterin was composed of inactivated Escherichia coli and Campylobacter fetus ssp. venerealis in a proprietary adjuvant. Tissues were collected for histopathology from 9 affected cattle that died or were euthanized after clinical signs lasting 6-38 days. A range of tissues, including the injection site lesion and lumbar spinal nerve roots, was obtained for ultrastructural examination from a cow with paresis of 31 days duration. There was locally extensive pyogranulomatous myositis with fibrosis and necrosis in right M. longissimus lumborum. Extension of the lesion into the vertebral canal via spinal nerve foramina resulted in focal pyogranulomatous inflammation in epidural fat and in adjacent dura mater. There was axonal degeneration in dorsal, lateral, and ventral columns and chromatolysis of spinal motor neurons in lumbar spinal cord, secondary to compression. A distinctive histologic and ultrastructural feature of pyogranulomata was the presence of osmiophilic material at the center of inflammatory foci, surrounded by macrophages and giant cells that contained intracytoplasmic lipid droplets. Ultrastructural examination of entrapped spinal nerves revealed axonal degeneration and loss of myelinated and unmyelinated fibers, segmental demyelination with remyelination, axonal spheroid formation, and early axonal regeneration.(ABSTRACT TRUNCATED AT 250 WORDS)

Mucolipidosis IV

Mucolipidosis Type IV is a rare, autosomal recessive disorder characterized by corneal opacification, mental retardation, and delayed motor milestones. Whereas lysosomal storage material has been demonstrated in biopsied tissues and leukocytes, the complete autopsy pathology, including neuropathology, is unknown. The metabolic defect remains speculative. We report the general and neuropathologic findings of the only known autopsy. In the central nervous system, neuronal loss in the cerebral cortex, basal ganglia, deep cerebellar nuclei, and brainstem nuclei was marked by astrocytosis; the cytoplasm of residual neurons had brown granules. These granules were positive with periodic acid-Schiff, Concanavalia ensiformis, and Sudan black, but not with Luxol-fast blue. Ultrastructurally, neurons contained lysosomes laden with osmiophilic, amorphous and granular material, and few lamellated membrane structures. Hepatocytes, epithelia, endothelia, chondrocytes, and tissue macrophages also stained positively with Datura stramonium and Ricinus communis-I agglutinins, with renal glomeruli also staining with peanut agglutinin; most non-neural cells contained osmiophilic granules on toluidine blue-stained, plastic embedded sections, corresponding to lamellated membrane structures. These findings complement the previously reported ocular morphology and brain and liver biochemistry performed in the same patient, and suggest that the storage material in neurons differs from that in non-neural cells. Furthermore, the underlying defect is not likely to be a deficiency of a single enzyme (i.e. a lysosomal hydrolase).

Acrodermatitis chronica atrophicans: A light and electron microscopic study

Degeneration of the elastica and collagen fibres in skin biopsies from patients with acrodermatitis chronica atrophicans was studied with light and electron microscopy. Elastic fibres were involved in the infiltrative stage while the elastin plexus was still present. In the atrophic phase, only fragments of elastic and oxytalan fibres were seen and the elaunin plexus was absent. Some collagen fibres were surrounded by osmiophilic material. In all biopsies, myelin sheaths were collapsed without axon structures. Spirochetes could be demonstrated in 69% of the biopsies and were most numerous in infiltrative and nodular lesions. The loss of elasticity of the skin in the atrophic phase may be caused by the destruction of both elastic and elastin fibres.

Asplenium trichomanesssp.trichomanesgametophyte. A light and electron microscope study

SUMMARYIn controlled culture conditions Asplenium trichomanes L. ssp. trichomanes spores germinate 8–10 days after sowing. The first protonemal cell and the primary rhizoid arise from two unequal divisions of the original spore cell. First an unbranched 5–6-celled germ forms which later becomes a spatula-, racket- and finally heart-shaped, bisexual prothallus with archegonia consisting of four rows of four cells and antheridia consisting of opercular, ring and basal cells. An additional notch with associated archegonia and antheridia develops in several-month-old gametophytes. The ultrastructure of the mature gametophyte is characterized by pleomorphic mitochondria, plastids which may be chloroamyloplasts, vacuoles containing osmiophilic materials and poorly developed endoplasmic reticulum of smooth type. The walls are crossed by many plasmodesmata. (Membrane) structures are visible inside the wall materials nearest the plasmalemma.

Lysosomal changes in rat spinal ganglia neurons after prolonged treatment with cisplatin.

We performed morphometric and ultrastructural studies to determine the morphological response of rat spinal ganglion sensory neurons to prolonged administration of cisplatin up to a total dose of 18 mg/kg. We quantitated the different types of lysosomal system (LS) bodies present (primary and secondary lysosomes, lipofuscin granules) as well as multivesicular bodies in treated and control animals. Five rats were examined per group. This ultrastructural study on cisplatin-induced changes in LS of spinal ganglia neurons shows that the total area and total number of LS structures are significantly increased by cisplatin treatment. The main specific changes were increase in number of small-size lysosomes and increase in number of polymeric lipofuscin granules. Other alterations observed were presence of nucleolar segregation, patches of neurofilaments and deposits of osmiophilic material in the perikaryon and axon hillock, all indicating that sensory neurons are a major target of cisplatin.

Early morphological changes of primary afferent neurons and their processes in newborn mice after treatment with capsaicin.

Degenerating figures of dorsal root ganglion (DRG) neurons and their central and peripheral processes (dorsal root and saphenous nerve) and terminals (central terminals in the superficial dorsal horn and cutaneous nerve of the hind paw dorsal skin) of neonatal mice were examined 30 min, 1, 2 and 5 h, and 2, 3, 5, and 10 days after subcutaneous injection of capsaicin on post-natal day 2. Many small DRG neurons showed degeneration 1 h after treatment. Scarcely any features of degeneration were seen in the DRG and dorsal root 10 days after treatment. The degenerating aspects of terminal axons in the marginal layer of the superficial dorsal horn were characterized by enlarged round axons with closely packed osmiophilic materials, lamellar bodies, and loss of axoplasmic organelles. Two types of central terminals (C-terminals) showed degeneration in the substantia gelatinosa from 30 min after treatment onward. One type consisted of small, round, sinuous or slender dark terminals (CI-terminals), and the other of large, pale, round or angular terminals (CII-terminals). Those that degenerated markedly had homogeneously electron-dense axoplasm with dilated synaptic vesicles and inclusion bodies. Extensive degeneration of terminal axons in the marginal layer occurred 5 h after treatment, whereas conspicuous degeneration of C-terminals occurred from 30 min to 10 days after treatment in the substantia gelatinosa. CI-terminals showed marked degeneration during the first 3 days, whereas marked degeneration of CII-terminals occurred between 5 and 10 days after treatment. This time difference between the peaks of degeneration of CI- and CII-terminals indicates an important difference in the origins of these two types of capsaicin-sensitive, nociceptive fibers in the superficial dorsal horn; CI-terminals are derived from small DRG cells, whereas CII-terminals are derived from larger DRG cells. Unmyelinated axons in the dorsal root, saphenous nerve, and dorsal skin of the hind paw showed similar degeneration patterns 2 h after treatment to those of terminal axons in the marginal layer. Thus, the degenerating profiles in the marginal layer suggest that these axons arose from collaterals of unmyelinated primary axons descending or ascending within the marginal layer. Numerous enlarged degenerating axons showing vacuolation were conspicuous in the dorsal skin 3 days after treatment. The synchronous degeneration of the smaller DRG neurons, their central and peripheral processes, and their CI-terminals in the substantia gelatinosa supports the idea that the smaller DRG neurons are directly influenced by capsaicin, and that their degeneration is followed by centrifugal degeneration.

Hormones regulating structural changes in the adipocytes of the female earwig Labidura riparia

Changes in periovarian fat body fine structure occur during the reproductive cycle of the female earwig Labidura riparia. During the process of vitellogenesis, high levels of protein synthesis are seen in the adipocytes. Simultaneously, the formerly osmiophilic lipid droplets become non-osmiophilic, while an osmiophilic material diffuses from lipid droplets to the cisternae of the rough endoplasmic reticulum (RER). These structural features alter during non-vitellogenic periods: RER and Golgi apparatus regress and seem to be inactive. Lipid droplets are once again osmiophilic and no material enters into RER cisternae. Using microsurgical manipulations, hormonal treatments and light and electron microscopy, we have investigated the regulation of these changes. The neuroendocrine cerebral centre pars intercerebralis, by the action of the juvenile hormone of the corpus allatum. triggers protein metabolism. Another neuroendocrine center, the pars lateralis. and the hormone 20-hydroxyecdysone, regulate structural lipid droplet changes in the adipocytes.

Ultrastructure of the Human Skeletal Muscle Sarcocyst

The ultrastructure of the human skeletal muscle sarcocyst found in Malaysia is reported. Sarcocyst-positive, formalin-fixed tongue tissues were postfixed in osmium tetroxide. The primary cyst wall consisted of a thin membrane supported by osmiophilic material that was interrupted regularly by vesicle-like invaginations. Although there were no cytophaneres, stubby protrusions of the primary wall were observed. These protrusions were accentuated by dense, curvilinear material externally. The primary wall was wavy over about half the cross section of the cyst. The granular ground substance underlying the primary wall occasionally contained hitherto undescribed coiled microtubular structures. Branching septa extended from the ground substance into the cyst, separating mature merozoites into compartments. A few peripheral metrocytes and many laminated myelin figure-like structures, probably degenerating merozoites, were found. Although the human muscular sarcocyst has the same basic ultrastructure as those found in other animals, the stubby protrusions and coiled microtubular structures in the ground substance have not been described previously in nonhuman animals.

Ultrastructure of the Interface between Erysiphe pisi and Its Nonhost Plant, Barley.

The interface between Erysiphe pisi and its nonhost, barley, was investigated by transmission electron microscopy. Coleoptiles of barley inoculated with the fungus were fixed at predetermined time intervals: (1) before penetration; (2) 30min after initiation of coleoptile cytoplasmic aggregation; (3) 15-16hr after cytoplasmic aggregation. When appressoria of the fungus matured on the surface of coleoptiles, the appressoria cell wall was thin and flat where the appressorium was in contact with the coleoptile surface. At this stage of development, no noteworthy changes occurred in coleoptile cells below appressoria. In specimens fixed 30min after the initiation of cytoplasmic aggregation, short penetration pegs from appressoria, breached the coleoptile cell wall and were surrounded by and embedded with papillae. The papillae at this stage were mostly electron-dense with some heterogeneous, electron-lucent regions. A large number of spherosomes appeared around the margin of papillae in cytoplasmic aggregates. Some of spherosomes were attached to the margin of papillae and probably allowed their contents to be deposited into the papillae. In specimens fixed 15-16hr after the initiation of cytoplasmic aggregation, prominent papillae were always found below appressoria which apparently had attempted to penetrate the cell wall of coleoptile. These papillae consisted of multiple, alternately electron-dense and -lucent layers. This suggests that osmiophilic and non-osmiophilic materials were deposited alternately during papilla development which occurred in a period of 15-16hr after the initiation of cytoplasmic aggregation. Thus, the gradual development of papillae probably depends on such a continuous deposition of materials.

Floral nectaries from Limodorum abortivum (L) Sw and Epipactis atropurpurea Rafin (Orchidaceae): ultrastructural changes in plastids during the secretory process

Ultrastructural features of the floral nectaries of 2 orchid species, limodorum abortivum (L) Sw and Epipactis atropurpurea Rafin, were compared. In particular, ultrastructural changes were followed in the plastids throughout the process of nectar secretion. There is evidence that plastids play an important role in this process. Before secretion begins, plastids of epidermal and subepidermal cells of the nectaries of both species, accumulated large quantities of starch. However, during the phase of nectar secretion, the plastids were found to contain little if any, starch, suggesting that starch degradation had augmented nectar sugar production. Plastoglobuli were evident during all developmental stages. The tubular reticulum found in plastids of pre-secretory nectaries was still evident during secretion, but was then found to contain an unknown, dense osmiophilic material

Resynthesis of Thylakoids and Functional Chloroplasts in the Desiccated Leaves of the Poikilochlorophyllous Plant Xerophyta scabrida upon Rehydration

Summary The ultrastructural changes in chloroplasts and other cell organelles in the desiccated, achlorophyllous leaves of Xerophyta scabrida, a poikilochlorophyllous desiccation tolerant (PDT) monocotyledonous plant, were examined during reconstitution of the photosynthetic apparatus after rehydration of airdried leaves. In the desiccoplasts (the former chloroplasts) of the air-dried leaves no thylakoids were present, only osmiophilic lipid material in the place of former grana and stroma thylakoids and groups of translucent plastoglobuli. Ten to 12 h after the start of the rehydration of air-dried leaves the resynthesis of chlorophylls and thylakoids began, fundamental structural changes occurred in desiccoplasts: the appearance of a small amount of starch, of primary thylakoids and of primary grana consisting of two appressed thylakoids, whereas the size of plastoglobuli descreased. At this stage the mitochondria appeared to be fully functional and to recover before the reconstitution of chloroplasts. Grana with 2–3 thylakoids were predominant 24 h after the start of rehydration; the degree of stacking and the ratio of appressed to non-appressed membranes increased. Translucent plastoglobuli were no longer seen, only much smaller osmiophilic plastoglobuli were visible. At 72 h after rehydration of air-dried leaves, grana of up to 7 thylakoids appeared, the degree of stacking increased further, starch granules became larger, as did the plastoglobuli, which also again turned translucent. The thylakoid system was then fully reconstituted and capable of ensuring the energy requirements of a normal rate of CO2 assimilation as well as synthesis and accumulation of excess lipids in the translucent plastoglobuli.

Late Infantile Ceroid-Lipofuscinoses

The aim of the present study is to investigate further the ultrastructural skin patterns in five cases of late infantile ceroid-lipofuscinosis: two of these were classic forms, the others were variants. Ultrastructural examinations of skin biopsies revealed the presence of characteristic cytosomes; typical lipofuscin, consisting of osmiophilic granular materials, curvilinear bodies, and fingerprint bodies. Different ultrastructural profiles were found simultaneously in each case, without a significant prevalence of any specific one, and were often associated with lipid droplets. These inclusions were found in several epidermal and dermal cells. A different degree of involvement of the myelinated sheaths in the five cases was observed. This difference could be genetically determined or perhaps related to different stages of the disease. The findings demonstrate the involvement of clinically unaffected skin and confirm the relevance of electron microscopic studies in diagnosing these disorders. In fact, recognition of typical ultrastructural changes is a valuable diagnostic tool that can be used in supplementing clinical and electrophysiological examinations, especially when the metabolic error is unknown and no diagnostic biochemical assay is available.

Endosperm Development in Solanum nigrum L. Formation and Distribution of Lipid Bodies

The formation and distribution pattern of lipid bodies was investigated in spring and summer-grown seeds of Solanum nigrum. L. Lipid bodies were first formed in endosperm cells abutting the young globular embryo. The initial distribution pattern resembled an inverted cone with the 'tail' of the cone pointed away from the embryo. As the seed developed, lipid body formation commenced in the endosperm cells immediately adjacent to the cone and then in the remaining endosperm cells. RER cisternae were frequently found appressed to one side of the lipid body and sometimes continuous with the electron-lucent zone which separated the electron dense osmiophilic core from the surrounding half-unit membrane. The electron-lucent zone was of variable width and more noticeable in spring-grown seeds. In summer-grown seeds only, the osmiophilic cores of adjacent lipid bodies were joined by bridges of similar osmiophilic material. When the cotyledons of the torpedo-stage embryo had curved (approx. 14 d after anthesis), the osmiophilic stainability of the lipid bodies began to disappear as they were converted into oleosomes. Glyoxysome-like organelles were found only in the endosperm's 'Zone of Separation and Secretion' and arose earlier in the spring-grown seeds than in the summer-grown seeds. The early formation of lipid bodies, the spatial and temporal deposition pattern and the occurrence of glyoxysome-like organelles only in the Zone of Separation and Secretion suggests that the lipid bodies act as a reserve to be utilized during periods of uneven or low transfer of photosynthetic products.

Correlative light and electron microscopy: Counting centrioles in individual sea urchin zygotes previously followed in vivo

Centrosomes are the ensembles of structures that define the poles of the mitotic apparatus. In animal cells, a centrosome typically consists of a pair of orthogonally arranged centrioles associated with osmiophilic pericentriolar material which nucleates the microtubules of the aster and central spindle. The precise doubling, or reproduction, of the centrosome during interphase is an important event in the cell’s preparations for mitosis.To characterize the mechanisms that control centrosome reproduction we need to follow the behavior of centrosomes within individual cells over several cell cycles. We use sea urchin zygotes as an experimental system because they have a rapid cell cycle, are large enough (100 um diameter) to easily manipulate, and are optically clear. However, the experiments pose two challenges. The first is to follow the behavior of centrosomes within individual zygotes for several hours with the light microscope and then recover just those individuals from a large population for ultrastructural analysis.

Development and Fine Structure of the Glandular Trichomes of Artemisia annua L.

Development of capitate glands on the leaves of annual wormwood (Artemisia annua L.) was monitored with scanning and transmission electron microscopy. Differentiation of foliar cells into gland cells began in the youngest leaf primordia. After differentiation into a 10-celled biseriate structure of two stalk cells, two basal cells, and three pairs of secretory cells, the cuticle of the six secretory cells separated from the cell walls to form a bilobed sac that eventually splits to release its contents. At every developmental stage, the cells of the the gland contained relatively little vacuolar volume. The secretory cells contained extensive endoplasmic reticulum. The plastids of each cell pair were different. At maturity, the apical cells contained proplastids or leucoplasts with only occasional thylakoids. The cell pair below the apical cell pair contained amorphous chloroplasts without starch grains. The basal cell pair contained proplastids or leucoplasts and the stalk cells contained chloroplasts. The stroma to thylakoid ratio in the secretory cell chloroplasts was high. Initially, osmiophilic product was observed most frequently associated with stacked thylakoids, plastid envelopes, and smooth endoplasmic reticulum, although it was associated with all cell membranes. Near the plasma membrane adjacent to cell walls bordering the subcuticular space, the cytoplasm was enriched in smooth endoplasmic reticulum containing osmiophilic material. The apical cell wall of the apical secretory cell pair was reticulated on the inner cytoplasmic side and contained osmiophilic staining on the cuticular side. During early senescence, osmiophilic product was commonly associated with outer mitochondrial membranes.

Ultrastructure of the vascular responses of cotton to Verticillium dahliae

Ultrastructural examination of cotton plants infected with Verticillium dahliae showed responses in the vascular tissue similar to those reported earlier in cotton infected with Fusarium oxysporum f.sp. vasinfectum. Contact cells reacted vigorously to infection, but the nature of the response depended on the location of the cells. The majority of the contact cells adjacent to initially infected vessels showed a reorganization of the cytoplasm with the formation of an apposition layer, a partitioning of the central vacuole into smaller vacuoles, and the appearance of osmiophilic material that was secreted through the plasmalemma and deposited in the apposition layer and in the pit regions of the vessels. The remainder of the contact cells adjacent to initially infected vessels contained a degenerated cytoplasm but no fungal hyphae. Contact cells adjacent to secondarily infected vessels responded with increased cytoplasmic activity and the direct secretion of osmiophilic material into the vessel lumens where it accumulated to form aggregates of various sizes and shapes that occluded the vessels. The fungus in these vessels was coated and embedded in this secreted material. Key words: contact cells, Gossypium hirsutum, Gossypium barbadense, vascular wilt, vessel occlusion.