Electron-dense Granules Research Articles

Juvenile recurrent parotitis: Soft foods contribute to the delayed development of salivary glands.

Juvenile recurrent parotitis (JRP) is the second-most common childhood disease of the salivary glands after mumps. Since popularisation of mumps vaccination, children suffered from JRP more often, and the aetiology remains unclear. Chinese children had the habit of soft foods due to the special dietary habit of Asia. To clarify whether mastication was related to the pathogenesis of JRP and whether the growth of salivary glands was influenced by soft diet. Investigation of dietary habit and masticatory efficiency from 2015 to 2018 of children diagnosed with JRP compared with the normal children by the dentition. Mice had been fed a soft diet beginning in their development phase. The gland weight, amount of saliva, salivary amylase, histological and ultrastructural observation and the expression levels of EGF, FGFr2 and Wnt3a had been tested. The JRP children preferred soft foods and had a significantly lower masticatory efficiency than do normal children. When normalised by body weight, the gland weight, amount of saliva and amount of salivary amylase in the experimental group were significantly lower. The ultrastructural results showed that the acinar cells in the experimental groups were smaller and contained fewer electron-dense secretory granules than those in the control groups. The expression levels of EGF, FGFr2 and Wnt3a in the salivary glands of mice in the experimental groups were significantly lower than those of mice in the control groups. The soft diet indeed influenced the salivary gland through insufficient mastication, which could be one of the primary factors inducing JRP.

Sperm characters of the aspidogastrean Rohdella amazonica (Aspidogastridae, Rohdellinae), a parasite of the banded puffer fish Colomesus psittacus.

The ultrastructural characteristics of the mature spermatozoon of the aspidogastrean Rohdella amazonica (Aspidogastridae, Rohdellinae) were studied by means of transmission electron microscopy. The sperm cell shows two axonemes of the 9 + '1' trepaxonematan pattern of Platyhelminthes, parallel cortical microtubules, a well-developed lateral expansion, external ornamentation of the plasma membrane, one mitochondrion, an electron-dense ring, a nucleus and granules of glycogen. The present results were compared with those observed in the aspidogastreans studied to date and in other Platyhelminthes. The lateral expansion and the electron-dense ring are typical characters for aspidogastreans. Although a lateral expansion has been described in other Platyhelminthes such as monogeneans and digeneans, the Aspidogastrea shows a much larger lateral expansion with both peripheral and internal microtubules. The dense ring is observed as a cylinder in a longitudinal view and shows a more granular appearance in sperm cells from the seminal vesicle in comparison to a more electron-dense appearance in sperm cells from the seminal uterine receptacle.

The interplay between Mn and Fe in Deinococcus radiodurans triggers cellular protection during paraquat-induced oxidative stress

The bacterium Deinococcus radiodurans is highly resistant to several stress conditions, such as radiation. According to several reports, manganese plays a crucial role in stress protection, and a high Mn/Fe ratio is essential in this process. However, mobilization of manganese and iron, and the role of DNA-binding-proteins-under-starved-conditions during oxidative-stress remained open questions. We used synchrotron-based X-ray fluorescence imaging at nano-resolution to follow element-relocalization upon stress, and its dependency on the presence of Dps proteins, using dps knockout mutants. We show that manganese, calcium, and phosphorus are mobilized from rich-element regions that resemble electron-dense granules towards the cytosol and the cellular membrane, in a Dps-dependent way. Moreover, iron delocalizes from the septum region to the cytoplasm affecting cell division, specifically in the septum formation. These mechanisms are orchestrated by Dps1 and Dps2, which play a crucial role in metal homeostasis, and are associated with the D. radiodurans tolerance against reactive oxygen species.

M6PR-Specific Targeting of Lysosomal Heparanase Regulates Platelet Hemostatic Function in Mice

Besides α-granules and dense granules, which play critical roles in and beyond hemostasis, circulating blood platelets and their precursor cells megakaryocytes contain lysosomes, the contents of which are also secreted during platelet activation. In their delivery to the lysosome, acid hydrolases bearing phosphomannosyl residues are trafficked from the trans-Golgi network to the acidic late-endosomal compartment via the mannose 6-phosphate receptor (M6PR) pathway. To determine the role of M6PR-specific targeting of lysosomal enzymes in platelet function, platelet parameters were investigated in M6pr-/- mice lacking the 46-kDa M6PR, the physiological role of which is unclear. M6pr-/- mice had normal platelet count but displayed an increased number of distinct proplatelet-like cells compared to control mice, as determined by immunofluorescent microscopy. Moreover, transmission electron microscopy revealed the presence of abnormal membrane tubulations, elongated and electron-dense granules, and large vacuole-like structures within resting M6pr-/- platelets. M6pr-/- platelets expressed normally major glycoproteins on their surface and von Willebrand factor and fibrinogen in their α-granules. M6pr-/- mice were hyper-thrombotic, as assessed by tail bleeding time, and M6pr-/- platelets adhered to type I collagen with a significantly greater propensity than control platelets under arterial shear in in vitro flow experiments. Heparanase, an endo-β-glucuronidase that cleaves extracellular matrix heparan sulfate proteoglycans, is the most abundant lysosomal enzyme in platelets. Thus, its contribution to the phenotype of M6pr-/- mice was investigated further. Heparanase expression was decreased in the bone marrow megakaryocytes and blood platelets of M6pr-/- mice and increased in M6pr-/- plasma, as evidenced by immunoblot and fluorescent microscopy analysis, consistent with its mistargeting in the absence of M6PR. Interestingly, pharmacological inhibition of heparanase with OGT 2115 normalized the adhesion of M6pr-/- platelets to collagen in vitro, indicating that increased plasma heparanase contributes to the thrombotic phenotype of M6pr-/- mice. Taken together, the data suggest that the M6PR-specific targeting of lysosomal heparanase plays a critical role in platelet function, thereby regulating hemostasis. Disclosures No relevant conflicts of interest to declare.

Shared and Distinctive Ultrastructural Abnormalities Expressed By Megakaryocytes in Bone Marrow and Spleen from Patients with Primary Myelofibrosis

Several evidences indicate that abnormal megakaryocyte (MK) maturation drives the development of Myelofibrosis (MF), the end stage of Philadelphia-negative Myeloproliferative neoplasms: a) presence in bone marrow (BM) of numerous clusters of MK with ultrastructural abnormalities indicating retarded maturation was the first cell hallmark reported for MF (Zucker-Franklin, Dahlem Workshop on Myelofibrosis-Osteosclerosis Syndrome Germany-West: Pergammon Press; 1975); b) studies in mouse models with MK-restricted JAK2V617F expression demonstrated that abnormal MK are necessary and sufficient to induce MF (Zhan, Leukemia 2016;30:23324) and c) both in patients and mouse models, the driver mutations induce a RSP14 ribosomopathy that reduces expression of GATA1 in MK, leading to retarded maturation (Vannucchi, AmJPathol 2005;167:849; Gilles, JCI 2017;127:1316). Due to fibrosis and hematopoietic failure in BM, MF patients develop hematopoiesis in spleen which also is rich of MK. Whether in MF MK in BM and spleen express similar ultrastructural abnormalities has not been investigated as yet. By transmission electron microscopy, we compared the ultrastructural features of MK from BM of 5 MF patients and spleen of 11 MF patients (9 JAK2V617Fhomo, 1 JAK2V617Fhomo/ASXl1R1068X, 2 JAK2V617Fhetero, 1 JAK2V617F hetero/ASXl1R693X, 1 CARLtyp1/TET2R1214W and 2 triple negative; 12 primary and 4 post-PV, IPSS score: low-intermedium 2, DIPSS score: intermedium 1-2). MF underwent splenectomy to reduce discomfort of large spleen size (>20 cm from the left costal margin). BM from 2 healthy controls (HC) and post-trauma spleen from 1 HC were analyzed as control. MK were numerous both in BM (43±5 vs 30±2/area in MF vs HC, p=0.027, Anova) and spleen (47±18 in MF vs below detection in HC). The majority (98%) of MK from MF BM had immature ultrastructural morphology while 59% of MK from HC BM were mature (p=0.00003). To a surprise, the majority (69%) of MK from MF spleen had also mature morphology with developed platelet territories. However, by contrast with the heavy electron dense granules found in the cytoplasm of HC MK, the cytoplasm of MK from both BM (56%) and spleen (32%) contained granules with light electron density, an indication of reduced protein content. MK from MF BM and spleen consistently expressed distinctive ultrastructural abnormalities: 100% of BM MK presented dilated nuclear membranes with abnormally large nuclear pores which in 33% of the cases were associated with thickening of the plasma membrane with extrusion of pseudopods and presence of glycogen droplets. By contrast, these abnormalities were not presented by spleen MK, 49% of which presented instead mitochondria with reduced crests. These results suggest that MK from BM and spleen express distinctive metabolic abnormalities: those from BM express insufficient lipid metabolism impairing membrane biosynthesis while those from spleen have impaired aerobic metabolism. As predicted by the impaired RSP14 signature reported in MF (Gilles, JCI 2017;127:1316), the cytoplasm of MK from MF BM contained reduced number of ribosomes (455±125 vs 1059±41 in MF vs HC, p=0.004) but the percent of ribosomes engaging with the rough endoplasmic reticulum (RER) was the same in both cases (41±2 vs 46±10%, p=0.80). The number of ribosomes was lower also in MK from spleen (157±85, p=0.0003 ) but in addition, MK from the spleen were poorly engaged with the RER (33±22%, p=0.008). These results indicate that in addition to low number of ribosomes resulting in defective mRNA translation, MK from spleen express ribosomes that engage poorly with RER leading to defective folding check and post-translational modification of nascent proteins. These results predict that partially different mechanisms drive the alterations of platelet territories observed in MK from BM and spleen in MF: defective membrane biosynthesis drives the paucity of the granules and the retarded maturation of the platelet territories observed in MK from BM. Insufficient proteins synthesis drives the poor protein content of granules found in MK both from BM and spleen but in addition the function of the proteins in the granules of MK from spleen may be impaired by improper protein folding check/post-translational modification. These results suggests lipid metabolism and RER functions as two potential novel therapeutic targets to restore MK function in MF. Disclosures Vannucchi: Incyte: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees; Italfarmaco: Membership on an entity's Board of Directors or advisory committees; CTI BioPharma: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Antifungal Effect of Saussurea lappa Roots Against Phytopathogenic Fungi and Resulting Morphological and Ultrastructural Changes

Phytopathogenic fungi infect plants during growth, development, and harvest and their products during postharvest storage and transport. Fungicides are applied indiscriminately to combat these pathogens, posing a serious threat to humans, animals, and the environment. Some filamentous fungi also produce mycotoxins, which pose further problems. In this study we evaluated the antifungal activity of Saussurea lappa root extracts against several potent phytopathogenic fungi (Fusarium solani, F. moniliforme, F. oxysporum, Helminthosporium sativum, and Macrophomina phaseoloni). Various solvent extracts (methanol, ethanol, dichloromethane, ethyl acetate, and water) of the S. lappa root were used. We found that methanol, ethanol, and dichloromethane extracts had potent antifungal activity against all tested isolates. M. phaseolina showed the highest inhibition (89%) with a 5% methanol extract of S. lappa, and severe damage to the morphology and ultrastructure of its cells was also observed. SEM micrographs revealed that cells treated with S. lappa extracts had distorted, peeled, twisted, and shortened mycelium and peeled, completely distorted, and underdeveloped conidia. Moreover, the dichloromethane S. lappa extract (5%) arrested conidial development completely. Similarly, TEM studies showed that treated cells lacked clear demarcations between the cell membrane and cell wall, and had heavy vacuolation, a detached cytoplasm, ruptured and distorted mycelium and conidia, and a clear deposition of electron dense granules on the outer surface. GC-MS and FTIR studies indicated the presence of important bioactive compounds and functional groups in the extracts. Our results indicate that S. lappa roots are a rich source of bioactive compounds that can affect the integrity, stability, and structure of the fungal cell membrane, ultimately leading to cell death. Thus, root extracts could serve as eco-friendly, safe alternatives to the fungicides that are applied at both the harvest and postharvest stages.

Cytoarchitectural improvement in Leydig cells of diabetic rats after treatment with aqueous and ethanol extracts of Dracaena arborea (Dracaenaceae)

Background and aimRecent studies have demonstrated the androgenic effects of Dracaena arborea in castrated and diabetic rats, but the cytoarchitectural mechanism at the level of Leydig cells (LCs) justifying this improvement in androgens production in diabetic rats has never been examined. We investigated the effects of aqueous and ethanol extracts of D. arborea on diabetes-induced cytoarchitectural impairments of LCs in rats. Experimental procedureBesides a normal group, 4 groups of diabetic rats were treated orally with Millipore water (10 ml/kg, diabetic), sildenafil citrate (1.44 mg/kg), aqueous (500 mg/kg) and ethanol (100 mg/kg) extracts of D. arborea for 21 days. On day 22, rats were sacrificed and the testes were removed and prepared for electron microscopic analyses of LCs ultrastructure. Results and conclusionThe ultrastructure of LCs in control rats was normal, while that in diabetic rats exhibited large heterochromatization in the nuclei, reduced amount of smooth endoplasmic reticulum with no lipid droplets in the cytoplasm, many autophagosomes and degenerated mitochondria containing lots of electron dense granules in the matrix. Interestingly, treatment with D. arborea especially its aqueous extract (500 mg/kg) alleviated these impairments, characterized by a rarification of heterochromatization in the nuclei coupled to an increase and the presence in the cytoplasm of prominent smooth endoplasmic reticulum and a reduction of electron dense granules in the matrix of mitochondria. These alleviating properties of D. arborea on LCs ultrastructure of diabetic rats could explain its androgenic potential. These results are useful for the management of patients suffering from diabetes-induced hypogonadism.

Morphology and ultrastructure of salivary glands of male treehopper <i>Tricentrus brunneus</i> Funkhouser (Hemiptera: Membracoidea)

The salivary glands of male treehopper Tricentrus brunneus Funkhouser comprise a pair of acinous principal glands each with an anterior lobe and a posterior lobe; a pair of elbow-shaped accessory glands. Every accessory gland connects with the principal gland via a thin accessory salivary duct. The anterior lobe contains three types of acini (I, II and III), whereas the posterior lobe possesses only one type of acinus (IV). Two efferent salivary ducts fuse into a common salivary duct. Different acinis cells are filled with secretory granules that are distinct in size, number, shape and electron-density, indicating a maturation process before secreted. Infoldings in different acini suggest dilution of substances before being secreted. The presence of microvilli in acinus IV and the accessory gland duct possibly indicates the undergoing maturation of secretions. Electron-dense fine granules existed in the accessory gland cells, possibly related to virus transmission.

Expression of endogenous phospholipase D1, localized in mouse submandibular gland, is greater in females and is suppressed by testosterone.

To clarify the signal transduction mechanism in the differentiation and secretion of salivary glandular cells, the present study was attempted to examine in the submandibular gland (SMG) of mice, the expression and localization of phospholipase D1 (PLD1), one of the important effector molecules working in response to the activation of intramembranous receptors by first messengers. In immunoblotting analysis, the expression of PLD1 was high at postnatal 4weeks (P4W) and decreased at P8W, and it was at negligible levels at newborn stage (P0W) and postnatal 2weeks (P2W). The expression of PLD1 was greater in females, and it was suppressed by administration of testosterone to female mice. In immuno-light microscopy, immunoreactivity for PLD1 at P4W was moderate to intense, in the forms of dots and globules mainly in the apical domains of immature granular convoluted tubule (GCT)-cells localized largely in the proximal portion of the female GCT. By P8W, it decreased in intensity and remained weak to moderate along the apical plasmalemma of cells throughout the course of the female GCT, whereas it was faint throughout the GCT of the male SMG at P4W and negligible at P8W. In immuno-electron microscopy, immature GCT-cells characterized by electron-lucent granules were immunoreactive and the immunoreactive materials were deposited close to, but not within, those granules. Typical GCT cells, characterized by electron-dense granules, were immunonegative. No significant immunoreaction for PLD1 was seen in acini of SMGs of either sex at any time point examined. It is suggested that PLD1 is involved in the signaling for secretion of immature GCT cells and influences differentiation of these cells, probably through their own secretory substances.

Ultrabright and Stable Luminescent Labels for Correlative Cathodoluminescence Electron Microscopy Bioimaging.

The mechanistic understanding of structure-function relationships in biological systems heavily relies on imaging. While fluorescence microscopy allows the study of specific proteins following their labeling with fluorophores, electron microscopy enables holistic ultrastructural analysis based on differences in electron density. To identify specific proteins in electron microscopy, immunogold labeling has become the method of choice. However, the distinction of immunogold-based protein labels from naturally occurring electron dense granules and the identification of several different proteins in the same sample remain challenging. Correlative cathodoluminescence electron microscopy (CCLEM) bioimaging has recently been suggested to provide an attractive alternative based on labels emitting characteristic light. While luminescence excitation by an electron beam enables subdiffraction imaging, structural damage to the sample by high-energy electrons has been identified as a potential obstacle. Here, we investigate the feasibility of various commonly used luminescent labels for CCLEM bioimaging. We demonstrate that organic fluorophores and semiconductor quantum dots suffer from a considerable loss of emission intensity, even when using moderate beam voltages (2 kV) and currents (0.4 nA). Rare-earth element-doped nanocrystals, in particular Y2O3:Tb3+ and YVO4:Bi3+,Eu3+ nanoparticles with green and orange-red emission, respectively, feature remarkably high brightness and stability in the CCLEM bioimaging setting. We further illustrate how these nanocrystals can be readily differentiated from morphologically similar naturally occurring dense granules based on optical emission, making them attractive nanoparticle core materials for molecular labeling and (multi)color CCLEM.

Metabolic activity of cells in the macula flava of the human vocal fold from the aspect of mitochondrial microstructure.

ObjectivesThere is growing evidence to suggest that the cells in the maculae flavae of the vocal fold mucosa are tissue stem cells of the human vocal fold. This study investigated the metabolic activity of the cells in the maculae flavae of the human vocal fold from the aspect of mitochondrial microstructure.Study DesignHistologic analysis of the human vocal folds.MethodsFive normal human adult vocal folds obtained from autopsy cases were investigated under transmission electron microscopy.ResultsMitochondria were randomly distributed in the cytoplasm of the cells. The morphological features of the mitochondria consisted of a double‐membrane‐bounded body containing matrices and a system of cristae. In each mitochondria, the lamellar cristae were sparse. The intercristal space was occupied by a mitochondrial matrix which contained electron‐dense matrix granules, mitochondrial DNA, and ribonucleoprotein granules. Some mitochondria spread out over or fused to the surface of a lipid droplet in the cytoplasm. In addition, both the mitochondrial outer and inner membranes and the membranes of the lipid droplets had disappeared. Some close association between mitochondria and rough endoplasmic reticulum was present.ConclusionsThe features of the mitochondria of the cells in the maculae flavae of the human vocal fold suggested that their metabolic activity and oxidative phosphorylation were low and that they may have shifted to the utilization of lipids to some extent for their metabolic needs.Level of EvidenceNA

Rapid Intramitochondrial Zn2+ Accumulation in CA1 Hippocampal Pyramidal Neurons After Transient Global Ischemia: A Possible Contributor to Mitochondrial Disruption and Cell Death

Mitochondrial Zn2+ accumulation, particularly in CA1 neurons, occurs after ischemia and likely contributes to mitochondrial dysfunction and subsequent neurodegeneration. However, the relationship between mitochondrial Zn2+ accumulation and their disruption has not been examined at the ultrastructural level in vivo. We employed a cardiac arrest model of transient global ischemia (TGI), combined with Timm's sulfide silver labeling, which inserts electron dense metallic silver granules at sites of labile Zn2+ accumulation, and used transmission electron microscopy (TEM) to examine subcellular loci of the Zn2+ accumulation. In line with prior studies, TGI-induced damage to CA1 was far greater than to CA3 pyramidal neurons, and was substantially progressive in the hours after reperfusion (being significantly greater after 4- than 1-hour recovery). Intriguingly, TEM examination of Timm's-stained sections revealed substantial Zn2+ accumulation in many postischemic CA1 mitochondria, which was strongly correlated with their swelling and disruption. Furthermore, paralleling the evolution of neuronal injury, both the number of mitochondria containing Zn2+ and the degree of their disruption were far greater at 4- than 1-hour recovery. These data provide the first direct characterization of Zn2+ accumulation in CA1 mitochondria after in vivo TGI, and support the idea that targeting these events could yield therapeutic benefits.

Ultrastructure of cytokine induced human natural killer cells

Background & Aim Natural Killer (NK) cells account for 3.66-26.74% of lymphocytes in the human peripheral blood. They secrete exosomes which express both NK-cell markers and cytokines. It has been shown that Interkin-2 (IL-2) stimulated the proliferation of natural killer cells, a sub-class of lymphocytes in human. In this study, the NK cells were isolated and cultured under different period of time, the ultrastructure of the highly purified activated NK cells were analyzed by transmission electron microscopy (TEM).The increase in the number of the NK cells were measured by flow cytometry. Methods, Results & Conclusion Methods The peripheral blood mononuclear cells (MNCs) were isolated by Ficoll centrifugation followed by phosphate buffer saline (PBS) with 10% Acid Citrate Dextrose (ACD). The MNCs were cultured according to the protocol of Cellex Natural Killer Cell Kit (Japan). The NK cells at Day 0, and cultured at Day 3, 5, 7 and 14 were sampled and fixed with 2.5% glutaraldehyde for 24 hours at 4°C, and processed for routine TEM examination. Samples at Day 0 and Day 14 were quantified by flow cytometry. Results The resting NK cell at Day 0 was about 3.5um with a round nucleus of about 1.7um in diameter. Electron-dense granules (EDGs) of varying sizes were observed (100-200nm) in figure 1. The NK cells started to proliferate after cytokine activation cultured at Day 3. The average size of the NK cells (6um) and nuclei (4um) increased in sizes when compared with Day 0. Membrane-bounded vesicles were observed at the proximity of Golgi area (Fig. 2). Electro-dense granules of similar sizes as at Day 0 were observed at Day 5. Micro-vesicles (150-200nm) and exosomes (50-100nm) were found associated with plasma membrane of NK cells at Day 7 (Fig. 3). The average size (280-330nm) and number of the EDGs increased at Day 14 when compared with Day 0 and Day 5 (Figs. 4). The average number of activated NK cells increased from 304 cells/ul at Day 0 to 1796 cells/ul at Day 14 as measured by flow cytometry. Conclusion An increase in number of NK cells after Interkin-2 (IL-2) activation was confirmed in the present study. With a better understanding of the characteristics of exosomes, micro-vesicles and electron-dense granules as observed in the activated NK cells is crucial as a new therapeutic tool in the future.

Biomarkers and the role of mast cells as facilitators of inflammation and fibrosis in chronic kidney disease.

Chronic kidney disease (CKD) is a clinical syndrome with many adverse sequelae and is currently a major global health and economic burden. Regardless of aetiology, inflammation and fibrosis are common manifestations of CKD. Unfortunately, the underlying pathophysiological mechanisms are poorly understood, and robust prognostic and early diagnostic biomarkers of CKD are lacking. One immune cell population that has received little attention in the context of CKD is mast cells (MCs). This mini review will examine the role of MCs as facilitators of kidney inflammation and fibrosis, propose a mechanistic structure for MCs in CKD, and give consideration to biomarkers specific for MC activation that can be deployed clinically. MCs are derived from haematopoietic stem cells. They are characterised by electron-dense granules in the cytoplasm, filled with preformed mediators. MCs can synthesise a range of bio-active compounds. Activation of MCs modulates an innate immune and adaptive effector response. Increased MC counts have been observed in animal models of kidney disease and a range of kidney diseases in humans where MC presence has been linked to biomarkers of kidney function and tissue damage. To further implicate MCs in CKD, several chemokines, cytokines and proteases released by MCs have been observed in their own right in various kidney diseases and linked to progressive CKD. One compound released by MCs that is of particular interest is the MC-specific protease tryptase. This protease is capable of activating the G-protein coupled receptor (GPCR) protease activated receptor-2 (PAR-2). PAR-2 is widely expressed throughout the kidney and highly expressed in the tubular epithelial cells where its activation induces robust inflammatory and fibrotic responses. Novel prognostic and diagnostic biomarkers of CKD are needed. MC-specific proteases [tryptase, chymase and carboxypeptidase A3 (CPA3)] are easily detectable in the blood but questionably in the urine. This review aims to promote these as prognostic and diagnostic biomarkers in the context of CKD.

The differential secretion of FSH and LH: regulation through genes, feedback and packaging

While the role of oestradiol and progesterone in the control of GnRH pulsatile secretion and generation of the preovulatory GnRH surge to induce release of the LH surge has been fully investigated, less attention has been given to changes in the pituitary gland that may sensitize gonadotrophs to switch from pulsatile release to surge release of LH, in particular. Furthermore, in the follicular phase while pulsatile secretion of LH is maximal, FSH secretion is reduced, yet both hormones are produced by the same gonadotrophs. The mechanisms whereby this differential release can occur are still unclear. The main regulator of FSH secretion is through the negative feedback effects of oestradiol and inhibin, which directly affect FSHbeta mRNA content and subsequent synthesis of FSH. FSH is then released predominantly via a constitutive pathway and the amount released is closely related to the rate of synthesis. In contrast, while basal LH secretion occurs via a constitutive pathway, the principal release of LH through pulsatile secretion is through the regulated pathway with GnRH stimulating the release of pre-synthesized LH contained in storage granules without significant changes in LHbeta mRNA. Secretogranin II (SgII) is associated with LH in these electron-dense storage granules and LH-SgII granules appear to be the principal form of granule released in response to GnRH through the regulated pathway. At the time of the preovulatory LH surge, granule movement to the gonadotrope cell membrane abutting a capillary, polarization, appears to play an important part in the priming mechanism for release of LH during the preovulatory LH surge in response to the GnRH surge. As there appears to be limited or no gonadotroph cell division in the adult pituitary gland, each gonadotroph passes through this synthesis and secretion pathway repeatedly through successive oestrous cycles. Packaging of LH and FSH into different secretory granules within the same cell is thus pivotal for the differential secretion of these gonadotrophins.

Histological and Ultrastructural Studies on the Intestine of Guntea Loach, Lepidocephalichthys Guntea (Cypriniformes, Cobitidae)

Abstract The cellular organizations of intestine in Lepidocephalichthys guntea (Hamilton, 1822) have been described by light as well as scanning and transmission electron microscopy. The intestine is short and straight like, marked into anterior, middle and posterior region based on mucosal folds, number and size of columnar epithelial cells and mucous cells, thickness of submucosa and muscularis layer. The mucosa of anterior intestine forms high folds, which are lined with compactly arranged columnar epithelial cells and mucous cells. In the middle intestine, folds are pointless whereas the posterior intestine is without folds. The submucosa is formed of thin layer of connective tissue, contained collagen bundles and blood capillaries, comparatively well developed in the posterior intestine. By scanning electron microscopy, outlines of the luminal surface of anterior and middle intestine is embossed with oval or rounded columnar epithelial cells contained densely packed stubby microridges. The posterior intestine has closely set longitudinal folds characterized with minute blood capillaries and columnar epithelial cells having inconspicuous microridges. Ultrastructurally, the mucosal surface of the intestine consists of mucous cells with electron dense granules and columnar epithelial cells having numerous microvilli, mitochondria, endoplasmic reticulum, lysosomes and Golgi body. Cellular components of the anterior and middle intestine participate in the absorption whereas the presence of enormous blood vessels and capillary net work of posterior intestine probably responsible for air breathing.

Development and characterization of a canine-specific anti-CD94 (KLRD-1) monoclonal antibody

Natural killer (NK) cells are non-T, non-B lymphocytes are part of the innate immune system and function without prior activation. The human NK cell surface determinant, CD94, plays a critical role in regulation of NK cell activity as a heterodimer with NKG2 subclasses. Canine NK cells are not as well defined as the human and murine equivalents, due in part to the paucity of reagents specific to cell surface markers. Canines possess NK/NKT cells that have similar morphological characteristics to those found in humans, yet little is known about their functional characteristics nor of cell surface expression of CD94. Here, we describe the development and function of a monoclonal antibody (mAb) to canine (ca) CD94. Freshly isolated canine CD94+ cells were CD3+/–, CD8+/–, CD4–, CD21–, CD5low, NKp46+, and were cytotoxic against a canine target cell line. Anti-caCD94 mAb proved useful in enriching NK/NKT cells from PBMC for expansion on CTAC feeder cells in the presence of IL-2 and IL-15. The cultured cells were highly cytolytic with co-expression of NKp46 and reduced expression of CD3. Transmission electron microscopy revealed expanded CD94+ lymphocytes were morphologically large granular lymphocytes with large electron dense granules. Anti-caCD94 (mAb) can serve to enrich NK/NKT cells from dog peripheral blood for ex vivo expansion for HCT and is a potentially valuable reagent for studying NK/NKT regulation in the dog.

Mesenchymal Stem Cells from Nucleus Pulposus and Neural Differentiation Potential: a Continuous Challenge.

Mesenchymal stem cells (MSCs) are well-characterized adult stem cells, recently isolated from human nucleus pulposus of degenerate and non-degenerate intervertebral disc. The attention to this source is linked to its embryologic history and cells may conserve a stronger aptitude to neuronal differentiation than other MSCs. Here, MSCs from nucleus pulposus (NP-MSCs) were successfully isolated and characterized for morphology, proliferation, and expression of selected genes. Subsequently, the neuronal differentiation was induced by 10days of culture with a neuronal medium. NP-MSCs subjected to neural differentiation media (NP-MSCs-N) showed a morphological and biochemical modifications. NP-MSCs-N displayed elongated shape with protrusion, intermediate filaments, microtubules, and electron dense granules and the ability to form neurospheres. Even if they expressed neural markers such as NESTIN, β-TUBULIN III, MAP-2, GAP-43, and ENOLASE-2, the neural differentiated cells did not show neither spontaneous nor evoked intracellular calcium variations compared to the undifferentiated cells, suggesting that cells do not have electric functional properties. Further studies are required in order to get a better understanding and characterization of NP-MSCs and analyzed the molecular mechanisms that regulate their neural differentiation potential.

Abstract 17188: Cryoem Analysis in Cardiac Isolated Mitochondria Reveals New Insights for CsA and mPTP Activation

Excessive Ca 2+ accumulation is the main source of cardiac tissue and cell death during myocardial ischemia-reperfusion injury (IR injury) and myocardial infarction. Calcium dysregulation and overload leads to mitochondrial dysfunction, excessive reactive oxygen species (ROS) production, catastrophic energy failure, and opening of the cyclosporine A-sensitive mitochondrial permeability transition pore (mPTP). Mitochondrial Ca 2+ accumulation also results in the formation of amorphous Ca 2+ -phosphate granules localized in the mitochondrial matrix. These amorphous electron-dense granules are main components of the mitochondrial Ca 2+ sequestration and buffering system by mechanisms not yet well understood. The two aims of the present study are to test the relationship of Ca 2+ -phosphate granule size and number in cardiac mitochondria 1) exposed to a bolus calcium sufficient to elicit permeabilization and 2) whether CsA-treated mitochondria alters granule formation and size. A time course series of CryoEM images was analyzed to follow the permeabilization process. CryoEM results showed that mitochondrial incubated for longer time-courses have increased number of small granules (40 - 110 nm), swelling, membrane rupture and induction of mPTP opening. Conversely, shorter incubation time resulted in less granules per mitochondrion yet of similar size (35 - 90 nm). CsA- treated mitochondria, on the other hand, showed bigger phosphate granules (120 - 160 nm), and both lower granules per mitochondria and mPTP opening susceptibility. These results suggest a novel mechanism for CsA in which Ca 2+ -phosphate granule sizes are enhanced while maintaining fewer per mitochondrion. This effect may explain why CsA-treated mitochondria have higher calcium tolerance, delayed Ca 2+ -dependent opening of the mPTP, and protects against reperfusion-induced myocardial necrosis.

The first ultrastructural observations of the egg-forming complex of Calicotyle affinis, an endoparasitic monocotylid monogenean which lacks a uterus.

This ultrastructural study of the female reproductive system of Calicotyle affinis, a monogenean without a uterus, demonstrates, for the first time in a monogenean, the presence of accessory cells located in the peripheral region of the ovary and a single large cell whose surface is penetrated by deep invaginations which restrict the passage of oocytes through the lumen of the distal extremity of the ovary. The cytoarchitecture of the epithelial lining of the two vaginae is a syncytium formed by an anucleate epithelial lining with sunken epithelial perykaria. The wall of the seminal receptacle is an enlarged prolongation of the vaginal epithelial lining. Fertilization occurs in the fertilization chamber and fertilized oocytes retain cortical granules within their cytoplasm, a characteristic known to occur in free-living flatworms. Our study also highlights concentrations of two distinct groups of Mehlis' gland cell ducts on either side of the proximal end of the ootype distinguished by ultrastructural characteristics of their secretory granules. The epithelial wall of the ootype is formed by a single layer of regular, columnar, glandular epithelial cells; these cells are closely adjacent, conjoined towards their apical region by septate junctions and produce rounded, electron-dense granules which are discharged into the ootype lumen via a merocrine or holocrine mechanism. Released granules concentrate around the eggshell and form an additional fibrous coat. The morphological diversity observed in the female reproductive system of the Monogenea is commented on it relation to clarifying patterns in monogenean evolution and for understanding the phylogeny of the Neodermata.