Histochemical demonstration of two subtypes of O-linked oligosaccharides in the rat gastric glands

The gastric glands synthesize glycoproteins whose oligosaccharides are linked to the peptide core mainly by the O-glycosidic bond, specifically removed by beta-elimination procedure. Our aim was to research the possibility of the existence of two subtypes of O-linked oligosaccharides with a different behavior to the removal procedure. The lectins from peanut (PNA) and Maackia amurensis (MAA-I) were histochemically used as markers of the O-linked oligosaccharides. Sections were also pretreated with beta-elimination and/or peptide N-Glycosidase F (PNGase-F) for the specific removal of O- and N-linked oligosaccharides, respectively. The lectin GNA, which mainly labels to N-linked oligosaccharides, was used to test the correct working of PNGase-F. To test the possibility that the beta-elimination treatment could remove the terminal sialic acid residues, the lectin LFA was used. The surface epithelium was negative to PNA, while it became strongly positive when beta-elimination was performed for 1 day. This staining was resistant to PNGase-F, suggesting that PNA was labeling to O-linked oligosaccharides. However, after beta-elimination for 5 days this staining is not observed. A similar pattern appeared with MAA-I. We propose the existence of two subtypes of O-linked oligosaccharides: labile and resistant. The labile O-linked oligosaccharides are removed with beta-elimination for 1 day, unmasking the PNA-positive oligosaccharides. These oligosaccharides are resistant O-linked oligosaccharides because staining is abolished with longer treatment of beta-elimination. The results with MAA-I also support this suggestion. In summary, the labile O-linked oligosaccharides are removed with short treatment, while the resistant O-linked oligosaccharides need a stronger procedure (for 5 days).     

Migration of human and mouse primordial germ cells and colonization of the developing ovary: an ultrastructural and cytochemical study

This review is an account of the origin and migratory events of primordial germ cells until their settlement in the gonad before sexual differentiation in the human as well as mice. In this context, the morphodynamic characteristics of the migration of the primordial germ cells, the macromolecular characteristics of the extracellular matrix of the migratory pathway, and the factors involved in the germ cell guidance have been analyzed and discussed in the light of recent advances in this field, by means of immunocytochemical procedures. The events prior to gonadal morphogenesis and the origin of the somatic cell content of the human gonadal primordium have been also analyzed. In particular, evidences are presented showing that cells derived from the coelomic epithelium and mesenchyme are at the origin of the somatic components of the gonadal primordium, and that a mesonephric cell contribution to the generation of somatic cell components of the genital ridge in humans should be discarded due to the morphological stability of the different nephric structures during the period preceding the sexual differentiation of the gonad.     

Structure elucidation of native N- and O-linked glycans by tandem mass spectrometry (tutorial)

Oligosaccharides play important roles in many biological processes. However, the structural elucidation of oligosaccharides remains a major challenge due to the complexities of their structures. Mass spectrometry provides a powerful method for determining oligosaccharide composition. Tandem mass spectrometry (MS) provides structural information with high sensitivity. Oligosaccharide structures differ from other polymers such as peptides because of the large number of linkage combinations and branching. This complexity makes the analysis of oligosaccharide unique from that of peptides. This tutorial addresses the issue of spectral interpretation of tandem MS under conditions of collision-induced dissociation (CID) and infrared multiphoton dissociation (IRMPD). The proper interpretation of tandem MS data can provide important structural information on different types of oligosaccharides including O- and N-linked.     

Photoreceptor cells in the Xenopus retina

This review summarizes our present state of knowledge about spectrally different photoreceptor cell types in the Xenopus retina. The classification of the photoreceptors was based on morphology, combined with immunolabelling with various anti-visual pigment antibodies and other molecular probes on semithin sections and retinal wholemounts. The majority of photoreceptors is represented by rods. Altogether 97-98% of the total rod population consists of the principal ("red") rods that are selectively labeled by N-terminal specific anti-bovine rhodopsin monoclonal antibodies (mAbs) and are maximally sensitive to green light. The other, rare, blue-sensitive rod type ("green rod") is thinner, not stained by these antibodies but binds C-terminal specific anti-rhodopsin mAbs. The major representatives of the cones are red-sensitive and consist of a morphologically heterogeneous group comprising both (principal and accessory) members of double cones, as well as large single cones. Outer segments in this group are selectively labeled by mAb COS-1, specific to the L/M group of cone visual pigments. Another, relatively rare cone type is similar in size, but slightly smaller than the large single cone and is not labeled by mAb COS-1. This cone type is assumed to have a blue-sensitive cone visual pigment. The third, least abundant, and immunocytochemically distinct cone type is a small single (miniature) cone, which binds mAb OS-2 relatively strongly, and anti-rhodopsin mAbs 4B4 and 1D4 weakly. By exclusion, this small single cone may be identical with the UV-sensitive cone. Further studies are needed, however, to identify the color sensitivity of the latter two cone types.     

Detection of haemoglobin in red cells of Xenopus laevis by immunofluorescent double labelling

Red cells from metamorphosing Xenopus laevis were treated with a fluorescein-conjugated guinea-pig antibody to Xenopus tadpole haemoglobin, a rabbit antibody to Xenopus adult haemoglobin, and a rhodamine-conjugated goat anti-rabbit globulin antibody. The presence of both red and green fluorescence in some cells indicated that at metamorphosis a proportion of the red cells contained both adult and tadpole haemoglobins.     

Role of alkyl moieties in the antiwear and antioxidant performance of alkyl phosphorodithioates

Phosphorodithioate derivatives having different alkyl substitu‐ents have been synthesised, while keeping the core phosphorus‐sulphur moiety intact. The change in antiwear, extreme‐pressure, and antioxidant properties with the variation in sub‐stituents at oxygen and at sulphur linkages in the phosphorodithioates has been studied. The role of the heteroatoms with regard to the alkyl substituents has been examined. The experimental data show that the chemical structure of an additive influences its physicochemical and tribological properties.     

Huge clusters of embryonic stem cells in human embryos: A morphologic study

Background: Nothing is known about huge clusters (HC) of embryonic stem cells (ESC) in human fetal organs (HFO). Aim: To know the status of HC‐ESC in HFO. Methods: Morphology and immunohistochemistry (IHC) in 32 HFO of 7–40 gestational weeks (GW). Results: HC‐ESC were seen in many HFO including central nervous system, spinal cords, spine, soft tissue, bone, skin, thyroid, lung, liver, pancreas, gall bladder, extrahepatic bile duct, adrenal, kidney, bladder, foregut, midgut, hindgut, female and male genital organs, and neurons. HC‐ESC's were composed of two populations depending on constituting cells. One were large cells with ample acidophilic cytoplasms with vesicular nuclei and nucleoli. The other were small cells with scant cytoplasm with hyperchromatic nuclei without nucleoli, resembling lymphocytes. The HC‐ESC were frequently showed neuronal differentiation. HC‐ESC were positive for NCAM, synaptophysin, NSE, chromogranin, PDGFRA, AFP, ErbB2, bcl‐2, KIT, MET. They were negative for CD45, CD3, CD20, EMA, CEA, CA19‐9, cytokeratin (CK) 7, CK8, CK18, CK19, MUC1, MUC2, MUC5AC, and MUC6. The mean Ki‐67 labeling index (LI) was 13% ± 7%. HC‐ESC showed a little glycogen but lacked mucins. These HC‐ESC were seen in 7–25 GW, and they were rarely seen in 26–40 GW. Conclusions: The morphology, IHC, and ontogeny of HC‐ESC were described. Microsc. Res. Tech. 77:825–831, 2014. © 2014 Wiley Periodicals, Inc.     

Symphony of rhythms in the Xenopus laevis retina

The photoreceptor layer in the retina of Xenopus laevis harbors a circadian clock. Many molecular components known to drive the molecular clock in other organisms have been identified in Xenopus, such as XClock, Xper2, and Xcrys, demonstrating phylogenetic conservation. This model system displays a wide array of rhythms, including melatonin release, ERG rhythms, and retinomotor movements, suggesting that the ocular clock is important for proper retinal function. A flow-through culture system allows measurements of retinal rhythms such as melatonin release in vitro over time from a single eyecup. This system is suited for pharmacological perturbations of the clock, and has led to important observations regarding the circadian control of melatonin release, the roles of light and dopamine as entraining agents, and the circadian mechanisms regulating retinomotor movements. The development of a transgenic technique in Xenopus allows precise and reliable molecular perturbations. Since it is possible to follow rhythms in eyecups obtained from adults or tadpoles, the combination of the flow-through culture system and the transgenic technique leads to the fast generation of transgenic tadpoles to monitor the effects of molecular perturbations on the clock.     

From egg to pole cells: ultrastructural aspects of early cleavage and germ cell determination in insects.

Insect eggs are giant and very complex cells covered by an extremely resistant shell. Both the egg cell and surrounding eggshell express anteroposterior and ventrodorsal polarity. The molecular and cytoplasmic organization of both axes originates during oogenesis and leads to the production of an ooplasmic system which consists of euplasm and deutoplasm (yolk) and contains a nucleus as well as extranuclear determinants of maternal origin. Both are part of the store of information for early embryogenesis. In addition, the deutoplasm serves as raw material and early nutrient supply for building the embryo. The insect egg cell, which is arrested in the first maturation division when released from the ovary during oviposition, will be activated by different stimuli among different species to complete meiosis and start embryogenesis. The zygote nucleus undergoes a number of synchronous mitotic divisions leading to cleavage energids which initially form a syncytial blastoderm and subsequently the cellular blastoderm. In many insects, prior to blastoderm formation, polar granules (or oosome material) are incorporated in a single cell or a small number of cells which bud off at the posterior pole. These so called pole cells give rise to the primordial germ cells. Therefore, polar granules or the oosome material mark the germ line, and while structural counterparts of determinants of body pattern formation have so far not been found, the polar granules or oosome serve as an autonomous ooplasmic determinant for the pole or germ cells. Anteroposterior body polarity can arise independent of the germ plasm.     

Identification of differential mRNA and lncRNA expression in AcMNPV-infected Sf9 cells

Sf9Sf9 are the ovarian cells of Spodoptera frugiperda that is the host of Autographa californica multiple nucleopolyhedrovirus (AcMNPV), and hence can serve as an effective test vehicle to understand the AcMNPV infection mechanism. In this study, through high-throughput sequencing technology using samples collected from Sf9 cells at different time points after AcMNPV infection, 3463 pieces of time-series differentially expressed RNA (1,200 mRNA and 2,263 lncRNA) are identified and justified by experimental verification of randomly selected samples from them, proving the validity of the bioinformatical analysis on this topic. Functional enrichment analysis and target prediction are performed on those differentially expressed RNA, from which the major functional enrichment distribution of those differentially expressed mRNA is derived. It has been found that the differential genes are mainly in the cellular anatomical entity and intracellular in terms of the cellular component, and in the binding and catalytic activity in terms of the molecular function. Also, the differential mRNA are mainly concentrated in global and overview maps, signal transduction, infectious diseases, and viral, etc. Moreover, those mRNA targeted by lncRNA are predicted. The correlation between those differentially expressed lncRNA and mRNA indicates that lncRNA is very likely playing an important role in the interaction between virus and host. Aided by an advanced co-expression analysis approach, the “hub” RNA is also identified. The study in this work pave the way for further analyzing and understanding how AcMNPV escapes from the host’s immunity, manipulates the host to realize the self-multiplication, and realizes the timely conversion between its two particle forms, laying the foundation for uncovering the host’s immune response process.     

Confocal microscopy and 3-D reconstruction of the cytoskeleton of Xenopus oocytes

Xenopus oocytes contain a complex cytoskeleton composed of three filament systems: (1) microtubules, composed of tubulin and at least three different microtubule-associated proteins (XMAPs); (2) microfilaments composed of actin and associated proteins; and (3) intermediate filaments, composed of keratins. For the past several years, we have used confocal immunofluorescence microscopy to characterize the organization of the oocyte cytoskeleton throughout the course of oogenesis. Together with computer-assisted reconstruction of the oocyte in three dimensions, confocal microscopy gives an unprecedented view of the assembly and reorganization of the cytoskeleton during oocyte growth and differentiation. Results of these studies, combined with the effects of cytoskeletal inhibitors, suggest that organization of the cytoskeleton in Xenopus oocytes is dependent upon a hierarchy of interactions between microtubules, microfilaments, and keratin filaments. This article presents a gallery of confocal images and 3-D reconstructions depicting the assembly and organization of the oocyte cytoskeleton during stages 0-VI of oogenesis, a discussion of the mechanisms that might regulate cytoskeletal organization during oogenesis, and speculates on the potential roles of the oocyte cytoskeleton during oogenesis and axis formation.     

Three-dimensional reconstruction of the remodeling of the systemic vasculature in early pig embryos

Current research on angiogenesis and vascular regression is mainly focused on pathological conditions such as tumor growth and diabetic retinopathy, while a suitable physiological model to study the controlling factors in these processes is still lacking. The remodeling pattern of the embryonic vasculature into the adult configuration, such as the branchial arch arterial system developing into the aorta or the early embryonic veins building the caudal vena cava can potentially serve as a model. However, practical applications of the embryonic vascular patterning are impeded by the current controversy over the exact development of the caudal vena cava in mammals. To elucidate these ambiguities, specific developmental stages of vascular development in pig embryos were mapped by means of computer-assisted 3D reconstructions starting from histological serial sections of Bouin's fixed embryos. Special attention was given to venous segments in the lumbar region, as their origin and fate are equivocally described in literature. Here we demonstrate that these venous segments originate from the caudal cardinal veins which are forced to migrate during development into a more dorsal position due to the expansion of the developing metanephroi and the more dorsal relocation of the umbilical arteries. These findings are in contrast with the generally accepted theory that the venous segments in the lumbar region arise from newly formed veins that are located dorsal to the early caudal cardinal system.     

Photoreceptor classes and transmission at the photoreceptor synapse in the retina of the clawed frog, Xenopus laevis

The photoreceptor population in Xenopus consists of a green-sensitive rod (lambda(max) = 523 nm), a blue-sensitive rod (lambda(max) = 445 nm) and three classes of cone. The largest cone is red-sensitive (lambda(max) = 611 nm). The intermediate cone is presumed to be blue-sensitive based on physiological criteria, whereas the miniature cone may be UV-sensitive. Horizontal cells (HC) are of two sorts: axon-bearing and axonless. The axon-bearing HC is of the luminosity type and probably contacts all types of photoreceptor. The axonless HC is of the chromaticity type and contacts only intermediate (blue) cones and at least one type of rod. During development dendrites of HCs and bipolar neurons penetrate photoreceptor bases. A progressive maturation of HC and bipolar synapses with rods and cones occurs between tadpoles stages 37/8 and 46. Neighboring rods and cones are joined by gap junctions. During this same period, the outer segments are laid down and photopigments synthesized. A linear relation was found between the quantum capturing ability of the rod and its absolute threshold. Mature rods of the Xenopus retina release glutamate in a calcium-dependent manner. Glutamate release was found to be a linear function of calcium influx through L-type calcium channels. Both types of HC possess ionotropic glutamate receptors of the AMPA subtype.     

Bones Morphogenic Protein‐4 and retinoic acid combined treatment comparative analysis for in vitro differentiation potential of murine mesenchymal stem cells derived from bone marrow and adipose tissue into germ cells

Nowadays, infertility is no longer considered as an unsolvable disorder due to progresses in germ cells derived from stem lineage with diverse origins. Technical and ethical challenges push researchers to investigate various tissue sources to approach more efficient gametes. The purpose of the current study is to investigate the efficacy of a combined medium, retinoic acid (RA) together with Bone Morphogenic Protein‐4 (BMP4), on differentiation of Bone Marrow Mesenchymal Stem Cells (BMMSCs) and adipose‐derived mesenchymal stem cells (ADMSCs) into germ cells. Murine MSCs were obtained from both Bone Marrow (BM) and Adipose Tissue (AT) samples and were analyzed for surface markers to get further verification of their nature. BMMSCs and ADMSCs were induced into osteogenic and adipogenic lineage cells respectively, to examine their multipotency. They were finally differentiated into germ cells using media enriched with BMP4 for 4 days followed by addition of RA for 7 days (11 days in total). Analyzing of differentiation potential of BMMSCs‐ and ADMSCs were performed via Immunofluorescence, Flowcytometry and Real time‐PCR techniques for germ cell‐specific markers (Mvh, Dazl, Stra8 and Scp3). Mesenchymal surface markers (CD90 and CD44) were expressed on both BMMSCs and ADMSCs, while endothelial and hematopoietic cell markers (CD31 and CD45) had no expression. Finally, all germ‐specific markers were expressed in both BM and AT. Although germ cells differentiated from ADMSCs showed faster growth and proliferation as well as easy collection, they significantly expressed germ‐specific markers lower than BMMSCs. This suggests stronger differentiation potential of murine BMMSCs than ADMSCs.     

Measurements of the diameters of the great arteries and semi-lunar valves of chick and mouse embryos

The great arteries of embryos are small channels of a complex three-dimensional arrangement. Measurements of their diameters, as required for understanding cardiovascular morphogenesis and the genesis of malformations, cannot be performed in two-dimensional histological sections. We present and evaluate a quick and simple method for performing highly significant and objective measurements of the diameters of blood vessels in vertebrate embryos and used this method for providing statistics of the diameter of the semi-lunar valves and the lumina of the great arteries of early chick and mouse foetus. We employed the high-resolution episcopic microscopy technique for generating volume data and three-dimensional computer models of the arterial trees of 30 chick embryos (Hamburger Hamilton stage 34), 30 mouse embryos of the OF1 strain harvested on 14.5 dpc, 30 embryos of the OF1 strain harvested on 15.5 dpc and 28 mouse embryos of the PARKES strain harvested on 14.5 dpc. The three-dimensional models (voxel size 2 μm × 2 μm × 2 μm and 3 μm × 3 μm × 3 μm) were used for defining virtual resection planes perpendicular to the longitudinal axis of the blood vessels at comparable positions. In these planes, we measured the lumen areas and the lumen perimeters. We also calculated the lumen diameter and the true lumen area from the perimeter and present statistical analysis. Finally, we evaluate and discuss the reliability and reproducibility of our method and present all measurements in a form that minimizes the influence of specimen size variation, specimen processing and data generation methods.     

Identification of tumorigenic risk genes in human placenta-derived mesenchymal stem cells treated with 3-methylcholanthrene

Mesenchymal stem cells (MSCs) capable of tumour topotaxis have been served as cellular vehicles to deliveranti-tumour agents. As cellular components of the tumour microenvironment, MSCs also affect tumour progression.However, the tumour transformation-related genes of MSCs remain unclear since either tumorigenic or tumoursuppressor effects within these cells have been researched. Hence, we aimed to identify potential biomarkers indicativeof tumorigenic risk by RNA-seq analysis of human placenta tissue-derived MSCs (hPTMSCs) exposed to thecarcinogenic agent, 3-methylcholanthrene (3-MC). Twenty-nine tumour transformation-related genes and threepluripotency-related genes were appraised as differentially expressed genes (DEGs) in hPTMSCs. Overexpression ofsfrp1 led to reduced cell viability, migration, and colony formation in A549. In contrast, the overexpression of ptgs2exerted the opposite effect. These results indicate that A549 cells with high ptgs2 expression but low sfrp1 expressionmay have a more potential tumorigenic capacity. Taken together, this study suggests that ptgs2 and sfrp1 may betumorigenic risk genes.