Perinuclear Area Of Cytoplasm Research Articles

P–648 In-vitro Evaluation of SCF and StxBP1 in Polycystic Ovary Syndrome (PCOS)

Abstract Study question Is the interaction between intrafollicular cells in PCOS, impaired by the change of vesicular fusion and/or exocytosis in granulosa cells (GCs)? Summary answer StxBP1 expression leves impared in GCs of PCOS. What is known already PCOS characterised as follicular arrest on antral follicles, cystic follicle formation, and follicular development failure. GCs secretes wide variety of factors via exocytosis, and plays critical role during folliculogenesis. Secretory vesicles are transported to cellular membrane. This process requires local concentrations of SNAREs consisting of tSNARE, vSNARE, and other vesicle fusion associated proteins. SNARE proteins are involved in vesicle fusion, exocytosis, and intracellular trafficking. GCs secretes KITL which provides follicular activation and growth. Syntaxins are one of the members of SNARE complex. StxBP1 is a protein which has a crucial role in secretory vesicle fusion that provides fusion of syntaxins. Study design, size, duration Granulosa cells (GCs) were collected for primary cell culture, since 2019 from both PCOS (n = 10) and healthy (male factor infertility) (n = 10) women undergoing ART. Each GCs from participant divided into two groups as in-vitro stimulated group and in-vitro nonstimulated group. Participants/materials, setting, methods GCs have been isolated from follicular fluid taken from patients during oocyte pick-up at Hacettepe University In-Vitro Fertilization Unit. nGCs were cultured at most secod subcultures after the isolation. The stimulated groups of both PCOS and control groups were stimulated by hCG(10IU/ml) ve FSH(0.5IU/ml) for 24 hours. Vesicle fusion proteins (Stx6, StxBP1, and SNAP25), KITL, and FSHr expressions were analyzed on granulosa cells from each group via immunofluorescent (IF) labeling and cyto-ELISA. Main results and the role of chance FSHr were compared in both control and PCOS before and after stimulation. There was no difference between FSHr expression levels in both groups. Indirect IF is widely considered for SNAP25, Stx6, StxBP1 proteins in all groups of GCs screening with/without stimulation. Expression of SNAP25, StxBP1 mainly scattered through all cytoplasmic area,s and membranous localization was observed. Stx6 expressions were particularly distinguished at perinuclear area of cytoplasm. However, stimulated cells of control appeared more peripherally Stx6 expression. This pattern caused by stimulation wasn’t observed in PCOS. Expressions of SNAP25, Stx6, StxBP1 were observed with less expression in PCOS. Also, the response to stimulation was lower than the control group. The differences in Stx6, SNAP25, StxBP1 and KITL levels before and after stimulation was evaluated for both control and PCOS in Cyto-ELISA. However, both SNAP25 and Stx6 expressions in GCs of both groups were similar in response to stimulation. The expression levels of StxBP1 in response to stimulation were significantly lesser than control at PCOS. KITL expressions were lower in the PCOS as expected furthermore similar to StxBP1 in response to stimulation. According to our findings, the highest response to stimulation in GCs occurred for StxBP1 and KITL in the control. Limitations, reasons for caution Since human cells were used in the study and the cells of each patient do not exhibit the same characteristics, the lowest number of patient samples identified in the statistical power analysis were included in the study. Wider implications of the findings: Our view to the disruption in the secretion of signal molecules in terms of vesicle dynamics will offer a new perspective in female infertility or cross-talks in folliclar cells of the ovary. Trial registration number TSA–2019–18196

Ginseng total saponin modulates podocyte p130Cas in diabetic condition

Proteinuric conditions demonstrate structural and compositional changes of the foot processes and slit diaphragms between podocytes. p130Cas in podocytes serves as an adapter protein anchoring glomerular basement membrane to actin filaments of podocyte cytoskeleton. To investigate the effect of ginseng total saponin (GTS) on the pathologic changes of podocyte p130Cas induced by diabetic conditions, we cultured mouse podocytes under: 1) normal glucose (5 mM, control); 2) high glucose (HG, 30 mM); 3) advanced glycosylation endproducts (AGE)-added; or 4) HG plus AGE-added conditions and treated with GTS. In confocal imaging, p130Cas colocalized with zonula occludens-1 and synaptopodin connecting to F-actin. However, diabetic conditions relocalized p130Cas molecules at perinuclear cytoplasmic area and reduced the intensity of p130Cas. In Western blotting, diabetic conditions, especially HG plus AGE-added condition, decreased cellular p130Cas protein levels at 24 and 48 h. GTS improved such quantitative and qualitative changes. These findings imply that HG and AGE have an influence on the redistribution and amount of p130Cas of podocytes, which can be reversed by GTS.

Ginseng total saponin regulates podocyte P-cadherin/β-catenin unit in diabetic condition

Proteinuric conditions demonstrate structural and compositional changes of the foot processes and slit diaphragms between podocytes. β-Catenin in podocytes serves as an adapter protein anchoring P-cadherin of slit diaphragms to actin filaments of podocyte cytoskeleton. To investigate the effect of ginseng total saponin (GTS) on the pathologic changes of podocyte P-cadherin/β-catenin unit induced by diabetic conditions, we cultured mouse podocytes under: 1) normal glucose (5 mM, = control); 2) high glucose (HG, 30 mM); 3) advanced glycosylation end products (AGE)-added; or 4) HG plus AGE-added conditions and treated with GTS. In confocal imaging, β-catenin colocalized with P-cadherin at intercellular junction area, predominantly. However, diabetic conditions relocalized and concentrated both molecules at perinuclear cytoplasmic area. In Western blotting, diabetic conditions, especially HG plus AGE-added condition, also decreased cellular β-catenin protein levels at 6, 24, and 48 hours. GTS improved such quantitative and qualitative changes of β-catenin. These findings imply that HG plus AGE have an influence on the redistribution and amount of P-cadherin/β-catenin unit of podocytes, which can be reversed by GTS.

Expression and characterization of UL16 gene from duck enteritis virus

BackgroundPrevious studies have indicated that the UL16 protein and its homologs from herpesvirus were conserved and played similar roles in viral DNA packaging, virion assembly, budding, and egress. However, there was no report on the UL16 gene product of duck enteritis virus (DEV). In this study, we analyzed the amino acid sequence of UL16 using bioinformatics tools and expressed in Escherichia coli Rosetta (DE3) induced by isopropy1-β-D-thiogalactopyranoside (IPTG). The recombinant protein was produced, purified using a Ni-NTA column and used to generate the polyclonal antibody against UL16. The intracellular distribution of the DEV UL16 product was carried out using indirect immunofluorescence assay.ResultsIn our study, UL16 gene of DEV was composed of 1089 nucleotides, which encoded 362 amino acids. Multiple sequence alignment suggested that the UL16 gene was highly conserved in herpesvirus family. The UL16 gene was cloned into a pET prokaryotic expression vector and transformed into Escherichia coli Rossetta (DE3) induced by IPTG. A 60kDa fusion protein band corresponding to the predicted size was produced on the SDS-PAGE, purified using a Ni-NTA column. Anti-UL16 polyclonal sera was prepared by immunizing rabbits, and reacted with a band in the IPTG induced cell lysates with an apparent molecular mass of 60 kDa. In vivo expression of the UL16 protein in DEV infected duck embryo fibroblast cells (DEFs) was localized mostly around perinuclear cytoplasmic area and in cytosol using indirect immunofluorescence assay.ConclusionsThe UL16 gene of DEV was successfully cloned, expressed and detected in DEV infected DEFs for the first time. The UL16 protein localized mostly around perinuclear cytoplasmic area and in cytosol in DEV infected DEFs. DEV UL16 shared high similarity with UL16 family members, indicating that DEV UL16 many has similar function with its homologs. All these results may provide some insight for further research about full characterizations and functions of the DEV UL16.

Effect of patulin on the interdigitating dendritic cells (IDCs) of rat thymus

Patulin is a common fungal contaminant of ripe apples used for the production of apple juice concentrates and it is also present in other fruits, vegetables and food products. Patulin is a secondary metabolite produced by species of the genera Penicillium, Aspergillus and Byssochlamys. Patulin has been reported to be mutagenic, carcinogenic and teratogenic. Antigen-presenting cells (APCs) are of prime importance in the innate immune response; they capture antigen in tissues and then migrate to the lymphoid organs to present the antigen to T lymphocytes. Thus, they are crucial for the initiation of immunity. Interdigitating dendritic cells (IDCs) are a subset of APCs that are present at the lymphatic organs. In the thymus, they act in positive and negative selection during T cell development. In the present study, patulin was administered orally to growing male rats aged 5-6 weeks. A dose of 0.1 mg kg(-1) bw day(-1) was given to rats for a period of 60 or 90 days daily. The effect of patulin on the IDCs of thymus was investigated by transmission electron microscopy (TEM), and the results were evaluated in terms of cell destruction. In the rats of the control group, it was observed that the IDCs had an indented nucleus, a clear cytoplasm and numerous membrane extensions. In the cytoplasm, a well-developed golgi complex, mitochondria, granular endoplasmic reticulum and a small number of lysosomal structures were observed. At day 60 of patulin-treated rat groups (P-60), loss of cristae in mitochondria and chromatin margination and lysis in the nucleus were found. It was observed that the IDCs had a perinuclear area of cytoplasm surrounded by a peripheral electron-lucent zone. In the cytoplasm of the 90-day patulin-treated rat group (P-90), a peripheral electron-lucent zone was also found, similar to the P-60 group. Additionally increase in vesicular and lysosomal structures, increase in apoptotic bodies and condensation of chromatin in the nucleus were noted. It was observed that patulin leads to apoptotic body formation and cell apoptosis in the IDCs of rat thymus especially in the P-90-treated groups.

Phosphatidylcholine-specific phospholipase C in mitogen-stimulated fibroblasts

To investigate expression, subcellular localization and mechanisms of translocation of phosphatidylcholine-specific phospholipase C (PC-PLC) during the cell proliferative response, biochemical, immunoblotting, and immunofluorescence analyses were performed on quiescent and mitogen-stimulated NIH-3T3 fibroblasts. Platelet-derived growth factor (PDGF), insulin and 12- O-tetradecanoylphorbol-13-acetate induced, in 10–60 min, PC-PLC translocation from a perinuclear cytoplasmic area to the plasma membrane. Following cell exposure to PDGF (60 min), the overall PC-PLC expression increased up to 2–3×, while the enzyme activity increased 5× in total cell lysates, 2× in the plasma membrane, and 4× in the nucleus; moreover, confocal laser scanning microscopy showed a progressive externalization of PC-PLC on the outer plasma membrane surface and its accumulation in the nuclear matrix. Pre-incubation of cells with the PC-PLC inhibitor tricyclodecan-9-yl potassium xanthate (D609), before PDGF-stimulation, not only reduced the enzyme activity in total cell lysates as well as in plasma membrane and nuclear fractions, but also blocked the mechanisms of PC-PLC subcellular redistribution. These effects were associated with a D609-induced long-lasting cell cycle block in G o.

Ultrastructure of cystocarp development in Gelidium robustum (Gelidiaceae: Gelidiales: Rhodophyta)

The ultrastructure of cystocarp development is described for the red alga Gelidium robustum (Gardn.) Hollenb and Abbott. The external cortical cells of the cystocarp remain uninucleate and unmodified, while the internal ones are binucleate, producing large mucilage sacs that discharge their contents into the cystocarpic cavity. Crystalline inclusions occur in both kinds of cells. Nutritive filaments, up to five cells long, are produced. Microtubules are associated with lipid droplets and occur in cells of the third-order filament, which probably function as food suppliers and food-conducting cells for the nutritive filaments. The younger, terminal cells of the nutritive filaments are electron-dense, having a large nucleus and lipid droplets and probably function as nutritive suppliers of the terminal gonimoblast cells. The older cells of the nutritive filaments are electron-light-possessing mucilage sacs, which function as mucilage suppliers of the cystocarpic cavity. Carpospore differentiation proceeds through three developmental stages. The youngest carpospores have an ovoid shape, possessing a large nucleus, proplastids with the peripheral thylakoid and some mucilage sacs, while they are surrounded by the carposporangial mucilage. Starch is polymerized in the perinuclear cytoplasmic area of the later stages of young carpospores. Intermediate-aged carpospores continue their starch polymerization and thylakoid development of plastids. Curved dictyosomes produce vesicles that contribute carpospore wall material. Mature carpospores have a nucleus, fully developed chloroplasts, numerous starch granules and adhesive vesicles, while they are surrounded by a two-layered wall and compressed mucilage, which constitutes a carposporangial wall.

Perinuclear accumulation of hepatitis A virus proteins, RNA, and particles and ultrastructural alterations in infected cells.

The exact intracellular site of hepatitis A virus (HAV) production is unknown, possibly due to its usually slow and inefficient replication. Using immunocytochemistry and in-situ RT-PCR, we show that in cells infected with the rapidly replicating HAV strain HAS-15, viral proteins and RNA are scattered throughout the cytoplasm and accumulate in the perinuclear cytoplasmic area. Various ultrastructural alterations were found in infected cells, such as large polyribosomes, swelling of the perinuclear space and the ER, and dilatation of Golgi cisternae. In addition, HAV infection induced the formation of large arrays of annulate lamellae. Direct visualization of HAV particles was scarce. The various ultrastructural alterations described here might represent different phases of the replicative cycle of HAV that is asynchronous in the infected cell layer.

Melanocytes and iris color. Electron microscopic findings.

To quantitatively associate iris color with melanocyte pigment content. Autopsy eyes were classified as uniform-blue, uniform-hazel, or uniform-brown or showing a darker peripupillary ring. Using electron microscopic images and computerized image analysis, area, number, and size of mature melanosomes within the perinuclear cytoplasmic area only or within perinuclear and peripheral cytoplasmic areas of the superficial stromal melanocytes combined were measured. Average melanosomal area per perinuclear cytoplasmic area (AMAC) and average number of melanosomes per perinuclear area (AMNC) significantly differed across iris color groups (overall P<.001). This result reflects the large difference between blue-uniform and all other color groups. A marginally significant (nominal) trend from blue-ring through brown-ring was also detected (P=.06 for AMAC and P=.07 for AMNC). The average perinuclear cytoplasmic area was larger in the central iris zone (within 1 mm around the pupillary margin) than in the intermediate iris zone (between 1 and 2 mm around the pupillary margin) (P=.002), but AMAC and AMNC did not significantly differ between zones. The average melanosome size did not differ significantly across color groups (P=.11). Differences in iris colors are at least partially attributed to variable AMNC and AMAC within superficial melanocytes.

ULTRASTRUCTURE OF CARPOSPOROGENESIS IN THE PARASITIC RED ALGA FAUCHEOCOLAX ATTENUATA SETCH. (RHODYMENIALES, RHODYMENIACEAE)

Carpospore differentiation in Faucheocolax attenuata Setch. can be separated into three developmental stages. Immediately after cleaving from the multinucleate gonimoblast cell, young carpospores are embedded within confluent mucilage produced by gonimoblast cells. These carpospores contain a large nucleus, few starch grains, concentric lamellae, as well as proplastids with a peripheral thylakoid and occasionally some internal (photosynthetic) thylakoids. Proplastids also contain concentric lamellar bodies. Mucilage with a reticulate fibrous substructure is formed within cytoplasmic concentric membranes, thus giving rise to mucilage sacs. Subsequently, these mucilage sacs release their contents, forming an initial reticulate deposition of carpospore wall material. Dictyosome vesicles with large, single dark‐staining granules also contribute to wall formation and may create a separating layer between the mucilage and carpospore wall. During the latter stages of young carpospores, starch is polymerized in the perinuclear cytoplasmic area and is in close contact with endoplasmic reticulum. Intermediate‐aged carpospores continue their starch polymerization. Dictyosomes deposit more wall material, in addition to forming fibrous vacuoles. Proplastids form thylakoids from concentric lamellar bodies. Mature carpospores are surrounded by a two‐layered carpospore wall. Cytoplasmic constituents include large floridean starch granules, peripheral fibrous vacuoles, mature chloroplasts and curved dictyosomes that produce cored vesicles which in turn are transformed into adhesive vesicles. Pit connections remain intact between carpospores but begin to degenerate. This degeneration appears to be mediated by microtubules.

Ultrastructure of Carposporogenesis in the Parasitic Red Alga Faucheocolax attenuata Setch. (Rhodymeniales, Rhodymeniaceae)

Carpospore differentiation in Faucheocolax attenuata Setch. can be separated into three developmental stages. Immediately after cleaving from the multinucleate gonimoblast cell, young carpospores are embedded within confluent mucilage produced by gonimoblast cells. These carpospores contain a large nucleus, few starch grains, concentric lamellae, as well as proplastids with a peripheral thylakoid and occasionally some internal (photosynthetic) thylakoids. Proplastids also contain concentric lamellar bodies. Mucilage with a reticulate fibrous substructure is formed within cytoplasmic concentric membranes, thus giving rise to mucilage sacs. Subsequently, these mucilage sacs release their contents, forming an initial reticulate deposition of carpospore wall material. Dictyosome vesicles with large, single dark-staining granules also contribute to wall formation and may create a separating layer between the mucilage and carpospore wall. During the latter stages of young carpospores, starch is polymerized in the perinuclear cytoplasmic area and is in close contact with endoplasmic reticulum. Intermediate-aged carpospores continue their starch polymerization. Dictyosomes deposit more wall material, in addition to forming fibrous vacuoles. Proplastids form thylakoids from concentric lamellar bodies. Mature carpospores are surrounded by a two-layered carpospore wall. Cytoplasmic constituents include large floridean starch granules, peripheral fibrous vacuoles, mature chloroplasts and curved dictyosomes that produce cored vesicles which in turn are transformed into adhesive vesicles. Pit connections remain intact between carpospores but begin to degenerate. This degeneration appears to be mediated by microtubules.

Ultrastructural study of interdigitating cells in the thymus of the spotless starling, Sturnus unicolor.

Interdigitating cells (IDC) in the thymus of the spotless starling, Sturnus unicolor, were examined by electron microscopy. They occur principally in the thymic medulla and corticomedullary border. They possess an irregular nucleus and a perinuclear area of cytoplasm, containing most of the membranous organelles, surrounded by a peripheral electron-lucent zone. Clusters of smooth Golgi vesicles and complicated labyrinthine membrane-membrane contacts are the most characteristic cytological features. Birbeck granules are absent. Lymphocytes, plasma cells and even myoid cells can be found embedded in the cytoplasm. Immature elements, intermediate between epithelial-reticular cells and interdigitating cells, are tentatively identified as prointerdigitating cells. The functional significance of IDCs, and their phylogenetic significance in the vertebrate immune system, is discussed.

Cytochemical demonstration of increased adenosine triphosphatase activity in lymphocytes activated in vitro by phytohaemagglutinin or by the mixed lymphocyte reaction.

Cytochemical investigations of ATPase activity were performed on lymphocytes isolated from peripheral blood and activated in vitro by phytohaemagglutinin or by the two-way mixed lymphocyte reaction. Uncultured lymphocytes showed very little activity localized in small granules. The activity increased markedly during transformation. In fully transformed and actively proliferating cells, the ATPase activity was intense and localized in a crescentic perinuclear area of cytoplasm which was pale-staining and vesicular in Giemsa-stained preparations. In mitotic cells, the activity was in discrete granules or elongated structures suggestive of mitochondria, scattered throughout the cytoplasm. The ATPase activity had a pH optimum of 8.5 to 9.5 and was strongly inhibited at pH 7.5. The activity was stimulated by Ca2+ and Mg2+ and was inhibited by p-chloromercuribenzoate but not by oligomycin, which appeared to enhance the reaction. Lead nitrate at a concentration of 3 mM did not inhibit the reaction.

Hormonal regulation of Sertoli cell differentiation.

This study was carried out to determine the possible hormone dependence of the postnatal changes on the cytological characteristics of the rat Sertoli cell. Neutralizing antisera to rat follicle stimulating hormone (AS/FSH) and sheep luteinizing hormone (AS/LH) were prepared in rabbits and made monospecific by suitable absorption. Groups of male Sprague-Dawley rats were injected from birth to 32 days with either AS/FSH, AS/LH, AS/LH + testosterone propionate (TP), or normal rabbit serum (NRS). In control 33-day-old animals, Sertoli cells displayed most of the fine structural characteristics of the adult type. Their nuclei were highly infolded and showed an elaborate shape. Euchromatin was largely predominant with a few heterochromatic masses associated with the nuclear envelope or with the prominent nucleolus. The perinuclear area of cytoplasm was particularly rich in mitochondria, smooth endoplasmic reticulum (SER) and Golgi complexes. Inter-Sertoli cell junctions were fully developed. After AS/LH treatment, the nuclei were reduced in size and adopted a smooth surface with very few infoldings. In addition, many clumps of heterochromatin were apparent close to the nuclear envelope and the number of the nucleoli was reduced. Sertoli cell cytoplasm was severely atrophied following AS/HL administration, showing sparse mitochondria, SER and Golgi profiles. Addition of TP to the AS/LH treatment failed to reverse these cytoplasmic changes but resulted in a reappearance of the membrane infoldings of the nuclear envelope and normal numbers of nucleoli. On the other hand, AS/FSH treatment did not induce any apparent morphological change in the Sertoli cell nuclei. The cell volume was reduced but most of the cytoplasmic organelles were apparently present in normal numbers. However, extensively dilated cisternae of the endoplasmic reticulum containing floccular material were a common and impressive change. No modifications were detected in the Sertoli cell junctions in any of the experimental groups. ABP testicular and epididymal levels were diminished in both AS/FSH and AS/LH treated groups, but the reduction was more marked in the latter. ABP levels remained low when TP was administered with the AS/LH. These observations indicate that in the absence of LH morphological differentiation and growth of Sertoli cells are severely inhibited and that FSH withdrawal causes specific changes in the ER that may indicate a trophic action on it. The failure of TP to reverse the effects of AS/LH administration could be attributed to the possibly severe FSH inhibition induced by the high testosterone plasma levels.