Granular Vacuoles Research Articles

Upregulation of Nrf2/HO-1 Signaling and Attenuation of Oxidative Stress, Inflammation, and Cell Death Mediate the Protective Effect of Apigenin against Cyclophosphamide Hepatotoxicity

Liver injury is among the adverse effects of the chemotherapeutic agent cyclophosphamide (CP). This study investigated the protective role of the flavone apigenin (API) against CP-induced liver damage, pointing to the involvement of Nrf2/HO-1 signaling. Rats were treated with API (20 and 40 mg/kg) for 15 days and received CP (150 mg/kg) on day 16. CP caused liver damage manifested by an elevation of transaminases, alkaline phosphatase (ALP), and lactate dehydrogenase (LDH), and histological alterations, including granular vacuolation, mononuclear cell infiltration, and hydropic changes. Hepatic reactive oxygen species (ROS), malondialdehyde (MDA), and nitric oxide (NO) were increased and glutathione (GSH) and antioxidant enzymes were decreased in CP-administered rats. CP upregulated the inflammatory markers NF-κB p65, TNF-α, IL-6, and iNOS, along with the pro-apoptotic Bax and caspase-3. Pre-treatment with API ameliorated circulating transaminases, ALP, and LDH, and prevented histopathological changes in CP-intoxicated rats. API suppressed ROS, MDA, NO, NF-κB p65, iNOS, inflammatory cytokines, oxidative DNA damage, Bax, and caspase-3 in CP-intoxicated rats. In addition, API enhanced hepatic antioxidants and Bcl-2 and boosted the Nrf2 and HO-1 mRNA abundance and protein. In conclusion, API is effective in preventing CP hepatotoxicity by attenuating oxidative stress, the inflammatory response, and apoptosis. The hepatoprotective efficacy of API was associated with the upregulation of Nrf2/HO-1 signaling.

CHANNELOPATHIES AND RELATED DISORDERS: EP.225 Autosomal dominant early onset vacuolar myopathy with granular material, without periodic paralysis, associated with c.1583C>A CACNA1S gene mutation

Dominant mutations in CACNA1S gene mainly causes hypokalemic periodic paralysis (PP)(hypoPP). We report a patient with progressive early onset myopathy associated with heterozygous CACNA1S mutation and aim to extend the description of phenotypical spectrum of CACNA1S-related muscle diseases. A 68-year-old man developed progressive proximal weakness from the age of 35 years. He had four siblings including an affected brother. His mother had PP and became wheelchair bound at 55 years. CK level was elevated (<1.5 upper normal value). Electroneuromyography showed axonal sensorimotor polyneuropathy and myogenic potentials. Thigh magnetic resonance imaging showed extensive muscle fatty replacement and muscle biopsy showed atrophic fibers with vacuoles containing granular material. Clinical exome identified the heterozygous c.1583G>A well-known pathogenic variant in CACNA1S. The patient's brother had PP from the age of 27. Neurological examination was normal at the age of 39. Muscle biopsy showed a vacuolar myopathy with granular material. No further medical information was available. The patient died at the age of 72 yrs. Molecular characterisation was not achieved. CACNA1S-HypoPP evolving to fixed myopathy in late adulthood is well described but isolated progressive myopathy was only exceptionally reported and never with an onset <50 years. We hereby report the earliest “myopathy onset” ever described, enlarging the phenotypical spectrum of CACNA1S-related myopathies and suggest considering it even in young adult-onset myopathies, especially when granular vacuoles are present.

Hydrallantois in cows naturally poisoned by Sida carpinifolia in Brazil.

Sida carpinifolia is a small subshrub that is distributed throughout Brazil and is responsible for lysosomal storage disease and occasional reproductive problems in cattle, goats, equids, sheep, and deer. We describe herein the clinical, epidemiologic, and pathologic features of hydrallantois in 3 cows naturally poisoned by S. carpinifolia in Rio Grande do Sul State, Brazil. Clinically, all cows had marked abdominal distension and mild ataxia. After natural death or euthanasia, autopsies revealed that the abdominal distension in all 3 cases was caused by severe enlargement of the uterus, which contained 100-120 L of translucent fluid within the allantois, in addition to adventitial placentation. Microscopic evaluation of the placenta revealed marked diffuse edema, sometimes with a myxomatous appearance. Neurons in the cerebellum and obex were swollen, with mild-to-moderate cytoplasmic granular vacuolation. Histochemical examination with lectins ConA, WGA, and sWGA revealed mild-to-marked staining in the cytoplasm of neurons of the cerebellum and medulla at the level of the obex, indicating the occurrence of α-mannosidosis.

Ultrastructural and cytogenetic analyses of mature human oocyte dysmorphisms with respect to clinical outcomes.

The study aimed to describe the ultrastructure of two human mature oocyte intracytoplasmic dysmorphisms, the bull-eye inclusion and the granular vacuole, with evaluation of clinical outcomes after intracytoplasmic sperm injection (ICSI) treatment. We retrospectively evaluated 4099 consecutive ICSI cycles during the period 2003-2013. Three groups were compared: controls, those with a bulls-eye inclusion, and those with granular vacuoles. Oocyte dysmorphisms were evaluated by transmission electron microscopy and in situ fluorescence hybridization (FISH). Detailed data on demographic and stimulation characteristics, as well as on embryological, clinical, and newborn outcomes, are fully presented. The bull-eye inclusion is a prominent smooth round structure containing trapped vesicles, being surrounded by lipid droplets. The presence of this dysmorphism in the oocyte cohort had no clinical impact except when transferred embryos were exclusively derived from dysmorphism oocytes. The granular vacuole is delimited by a discontinuous double membrane and contains lipid droplets and vesicles. As FISH analysis revealed the presence of chromosomes, they probably represent pyknotic nuclei. The presence of this dysmorphism in the oocyte cohort had no clinical impact except when at least one transferred embryo was derived from dimorphic oocytes. Poor clinical outcomes were observed with transfer of embryos derived from dysmorphism oocytes, although without causing gestation or newborn problems. The bull-eye inclusion and granular vacuoles may thus be new prognostic factors for clinical outcomes.

Ultrastructure and molecular cytogenetics of human metaphase II oocytes with granular vacuoles

AbstractStudies demonstrated poor fertilization rates and compromised early embryo development in cases of oocytes with dark and granular ooplasm, or with central granular ooplasm and presence of a refractile body. Many other structural abnormalities of human mature oocytes do exist, but remain to be evaluated. In the present study we characterized granular vacuoles (GVa) of mature metaphase II human oocytes. Surplus abnormal MII oocytes (n=6) with GVa were used under informed consent, after controlled superovulation during IVF treatment cycles. In all cases, patients had normal karyotypes. For electron microscopy oocytes were fixed with karnovsky fixative for 2h at 4°C, post-fixed in 1-2% OsO4 in buffer containing 0,8% K3Fe3+(CN)6, dehydrated in ethanol series, equilibrated with propylene oxide and embedded in Epon. Semithin sections were stained with aqueous azur II and methylene blue (1:1). Ultrathin sections were stained with 3% aquous uranyl acetate (20 min) and Reynols lead citrate (10 min). Sections were observed with a transmission electron microscope JEOL 100CXII operated at 60kV.

Clinical and Immunohistochemical Responses of Plantar Warts to Topical Immunotherapy with Diphenylcyclopropenone

A 30-year-old man with bilateral plantar warts of the mosaic type which had been resistant to standard treatment modalities was treated with diphenylcyclopropenone. After 10 weeks, the treated warts had disappeared; the untreated warts, although showing some involution, still persisted. The untreated warts, serving as a control to prove the effectiveness of topical immunotherapy, responded likewise to subsequent treatment with diphenylcyclopropenone. Wart regression was reflected histopathologically by decreases in acanthosis, papillomatosis, granular vacuolation, and hyperkeratosis. Immunohistochemically, Ki-67 expression was markedly reduced, and a reversal of the CD4/CD8 ratio was seen. These findings suggest a major role of a cell-mediated immune response in the spontaneous resolution of warts.

Ultrastructure of Carposporophyte Development in the Red Alga Gloiosiphonia verticillaris (Cryptonemiales, Gloiosiphoniaceae)

The ultrastructure of carposporophyte development is described for the red alga Gloiosiphonia verticillaris Farl. The auxiliary cell produces gonimoblast initials, which divide to produce two types of gonimoblast cells—the nondividing vacuolate cells and terminal generative gonimoblast cells. The generative gonimoblast cells form clusters of carpospore initials, which eventually differentiate into carpospores. After gonimoblast filaments are formed, the auxiliary cell undergoes autolysis, causing degeneration of septal plugs between the auxiliary cell and adjacent cells, thus forming a fusion cell. Since this cell lacks starch and appears degenerate throughout carposporophyte development, a nutritive function cannot be ascribed to the fusion cell. Carpospore differentiation is simple and proceeds through three developmental stages. Young carpospores structurally resemble gonimoblast cells, because they contain undeveloped plastids, large quantities of floridean starch, and are surrounded by extensive mucilage instead of a distinct wall. In addition, dictyosomes form and begin to produce vesicles with fibrous contents representing carpospore wall material. During the intermediate stage, dictyosomes continue to produce vesicles that contribute additional carpospore wall material, thereby compressing the mucilage and creating a darker-staining layer outside the carpospore wall. Plastids form internal thylakoids by invaginations of the inner membrane of the peripheral thylakoid. The endoplasmic reticulum forms large granular vacuoles that appear to be degraded during subsequent stages of development. Mature carpospores form cored vesicles. They also contain mature chloroplasts, large amounts of floridean starch, and occasionally granular vacuoles. During this stage, interconnecting carpospore-carpospore and carpospore-gonimoblast cell septal plugs begin to undergo degeneration. This process may be mediated by tubular structures.

ULTRASTRUCTURE OF CARPOSPOROPHYTE DEVELOPMENT IN THE RED ALGA GLOIOSIPHONIA VERTICILLARIS (CRYPTONEMIALES, GLOIOSIPHONIACEAE)

The ultrastructure of carposporophyte development is described for the red alga Gloiosiphonia verticillaris Farl. The auxiliary cell produces gonimoblast initials, which divide to produce two types of gonimoblast cells—the nondividing vacuolate cells and terminal generative gonimoblast cells. The generative gonimoblast cells form clusters of carpospore initials, which eventually differentiate into carpospores. After gonimoblast filaments are formed, the auxiliary cell undergoes autolysis, causing degeneration of septal plugs between the auxiliary cell and adjacent cells, thus forming a fusion cell. Since this cell lacks starch and appears degenerate throughout carposporophyte development, a nutritive function cannot be ascribed to the fusion cell. Carpospore differentiation is simple and proceeds through three developmental stages. Young carpospores structurally resemble gonimoblast cells, because they contain undeveloped plastids, large quantities of floridean starch, and are surrounded by extensive mucilage instead of a distinct wall. In addition, dictyosomes form and begin to produce vesicles with fibrous contents representing carpospore wall material. During the intermediate stage, dictyosomes continue to produce vesicles that contribute additional carpospore wall material, thereby compressing the mucilage and creating a darker‐staining layer outside the carpospore wall. Plastids form internal thylakoids by invaginations of the inner membrane of the peripheral thylakoid. The endoplasmic reticulum forms large granular vacuoles that appear to be degraded during subsequent stages of development. Mature carpospores form cored vesicles. They also contain mature chloroplasts, large amounts of floridean starch, and occasionally granular vacuoles. During this stage, interconnecting carpospore‐carpospore and carpospore‐gonimoblast cell septal plugs begin to undergo degeneration. This process may be mediated by tubular structures.

Shaking pups: a disorder of central myelination in the spaniel dog. II. Ultrastructural observations on the white matter of the cervical spinal cord.

The ultrastructure of the cervical cord is described in a new canine mutant with severe hypomyelination of the C.N.S. Axons were either non-myelinated or surrounded by a myelin sheath that was markedly reduced in both its thickness and length of internode. Myelinated and non-myelinated zones were present on a single axon. There was no paucity of oligodendrocytes but many of those present contained empty or granular vacuoles within the cytoplasm. Features suggesting immaturity of myelination were commonly found at paranodes and along the internode. Abnormal inter-relationships of oligodendrocytes and astrocytes were present at many paranodes. These observations suggest an intrinsic defect of oligodendrocyte metabolism such that they are incapable of normal extension of their plasma membranes, while the cytoplasmic vacuoles may represent breakdown of defective lipids.

Electron microscope studies of cyst stages of Sarcocystis tenella: the origin of micronemes and rhoptries.

The origin and the development of the micronemes and rhoptries was investigated at the ultrastructural level in cyst stages of Sarcocystis tenella. During endodyogeny the micronemes appear in small vesicles distributed at the periphery of two large granular vacuoles in each daughter cell. Later, these vacuoles were divided into numerous vesicular spiral formation-centers, producing micronemes at the apical pole of young merozoites. Rhoptries were observed to originate from desnifications with in the same large vacuoles which gave rise to the micronemes.

Electron microscopy of Golgi elements in sea urchin eggs

The ultrastructure of the Golgi element (dictyosome) in sea urchin oogenesis and development has been studied by means of electron microscopy in ultrathin sections prepared according to the technique of Sjöstrand. 1. 1. The dictyosome of the growing oocyte and the fertilized egg is a stack of aggregated flattened vesicles in approximately 7 parallel rows. A few vesicles of a dictyosome may be swollen. The vesicles are closed and do not anastomose. Generally, smaller vesicles are found at the edges of the dictyosome. This structural organisation of Golgi membranes, vacuoles and “granules” is similar to the Golgi apparatus from a vertebrate cell. These Golgi components have the same membrane thickness—60 Å. A Golgi ground substance is barely discerned. 2. 2. In the growing oocyte many dictyosomes have a break of all vesicles, which bisects them at the same level. This probably represents a divisional stage. Dictyosomes of all diameters—0.2 to 2.4μ—have approximately the same thickness—0.2μ. 3. 3. In the very young oocyte the dictyosomes are collected into a circular formation near the nucleus. The gastrula cell also has the dictyosomes in the Golgi zone, whereas the other stages studied have the dictyosomes scattered in the cytoplasm. The mature egg has only a few dictyosomes, and these are of relatively small diameter. 4. 4. It is not possible to stratify the dictyosomes within the sea urchin egg by centrifugation. The classical method of visualizing the Golgi apparatus by osmium impregnation did not result in specific staining. 5. 5. Some dictyosome vacuoles of a growing oocyte contain a granular component. Most morphological stages from the granular vacuoles to yolk granules can be found, which suggests that yolk granules may originate in the dictyosomes. 6. 6. In the mature egg there appears a cell component consisting of a stack of flattened, closed vesicles in approximately 7 parallel rows. The thickness of the membrane is 60 Å; the total thickness of the vesicle, where its sides are parallel, is approximately 240 Å. The vesicles are not aggregated, and the total size of the component ( c. 3 × 0.5 μ) is larger than that of an aggregated dictyosome. This formation has been called a “dispersed dictyosome”. 7. 7. A dictyosome is found at the base of the flagellum in the flagellated cells of a gastrula.

The Division of the Macrospore Nucleus

1. In Lilium Philadelphicum the archesporial cell develops directly into the macrospore, and its nucleus during the first-division appears with twelve chromosomes, or half the number which are present in the vegetative nuclei. At quite an early stage in the development of the macrospore the linin thread of the chromatin network begins to thicken, and the chromatin granules undergo transverse fission. After division of the chromatin granules the whole chromatin band undergoes longitudinal splitting, and the double threads thus produced begin to twist upon each other. This twisted band finally manifests itself as a single continuous spirem, which doubles up and twists into twelve loops. The twelve loops break apart and give rise to the twelve chromosomes. The two linin threads with their granules, which compose the loop, continually become more intimately associated, so that the loop appears like a single linin thread with two irregular rows of chromatin granules. The chromatin loops become shorter by contraction, and receive a thick deposit of some substance which stains light at first but later takes the same color as the chromatin, giving the chromosomes the appearance of homogeneous, somewhat irregularly bean shaped bodies, in which the original chromatin loop is rarely visible. The chromosomes arrange themselves in the equatorial plane in such a manner that the end containing the two free ends of the original chromatin loop is in contact with the spindle fibers. The fibers gradually pull the chromosomes apart in such a manner that the original chromatin loop is untwisted and finally cut in two by a transverse division. Thus the parts of each chromosome which pass to the daughter nuclei represent transverse halves of the original chromatin loop, formed from about one twelfth of the double spirem. 2. The nucleus at first usually has about three nucleoli, each with one or more large granular vacuoles. After the longitudinal splitting of the chromatin band there arise in the nuclei numerous small vacuolate bodies. These are successively abstricted from the mother nucleolus by a process of budding, and give rise to numerous micronucleoli, which all pass out into the cytoplasm before the formation of the mother star, and later, at about the beginning of the close daughter skeins, these micronucleoli all pass back into the daughter nuclei, and by aggregation form the new nucleoli of the daughter nuclei. This process is repeated for every division of the female gametophyte. 3. At about the time of the division of the chromatin granules, there appear in the cytoplasm peculiar cytoplasmic threads, which pass from one side of the cell to the other, and are mostly tangent to the nucleus. At a later stage, at about the beginning of the nucleolar migration, these threads have disappeared, and numerous radiating threads pass out at right angles from the nuclear surface and extend to the cell walls. These radiations seem to hold some relation to the migration of the micronucleoli. Similar radiations appear around the daughter nuclei, and the micronucleoli, as they are drawn into the daughter nuclei, seem to be in contact with these cytoplasmic threads. 4. Two centrospheres appear beside the resting nucleus, and in the mother star stage a single centrosphere appears at each pole of the spindle; while a little later, during metakinesis, a centrosphere appears at each point with a double centrosome. In the daughter skein stage there are two centrospheres at each pole, which are often quite distinct and can easily be differentiated from the micronucleoli.