Immunocytochemical evidence for intragranular processing of pro-opiomelanocortin in the melanotropic cells of the rabbit

Summary The immunogold technique, employing antisera with clear-cut specificities, was used to localise different processing stages of pro-opiomelanocortin (POMC) in rabbit melanotropic cells. While the antiserum against ₃-MSH labelled all the secretory granules including intrasaccular condensations in the Golgi apparatus, antisera against -MSH only labelled extra-Golgi secretory vesicles (SV). All extra-Golgi SV were likewise labelled with the three antisera against -MSH used, despite their different specificities for the desacetylated, N-acetylated or C-amidated forms of the peptide. The antibody against -endorphin also labelled the extra-Golgi SV, while only some SV were labelled with the antibody against -endorphin. These results correlate with biochemical data in favour of mainly — if not exclusively — intragranular processing of POMC. Except for ₃-MSH, the cleavage of which could coincide with Golgi packaging of secretory material, other post-translational modifications of the precursor seem to occur when SV are discharged outside the Golgi area. The cleavage of -endorphin appears to be a later step in POMC processing, occurring in some mature SV.     

Exocytosis of glycogen during maturation of amphibian oocytes

The repartition and fate of glycogen β has been followed during progesterone-induced maturation of amphibian oocytes. The use of specific staining, both at the cytological and ultrastructural level, demonstrates that glycogen tends to be extruded from the oocyte during maturation of the urodeles Pleurodeles waltlii and Ambystoma mexicanum. No such effect of the hormone is observed in Xenopus laevis, where only a slight centrifuge migration of the glycogen could be recorded. Stacks of annulate lamellae increase during the early phase of in vitro progesterone-induced maturation (2 to 9 hours after progesterone application). After germinal vesicle breakdown (about 12 hours after beginning the progesterone treatment) annulate lamellae have disappeared and numerous masses of vesicles are present in the cytoplasm of Pleurodeles and Ambystoma matured oocytes. We never observed any close relation between the annulate lamellae and these vesicles.     

Further characterization of boar sperm plasma membrane proteins with affinity for the porcine zona pellucida

Boar sperm plasma membrane proteins (PMPs) with affinity for the zona pellucida were partially purified from columns of dextran sulfate using a linear salt gradient and a buffered detergent that retained their ability to block directly the binding of uncapacitated and capacitated sperm to isolated porcine oocytes. PMPs that bound most strongly to dextran sulfate (fraction IV) were also most effective in blocking sperm binding to porcine oocytes. These tightly bound proteins also bound to isolated zonae to a greater extent than other fractions. Monovalent antibodies to fraction IV PMPs completely blocked sperm binding to isolated eggs. Fraction IV PMPs lost the ability to inhibit directly the binding to eggs when treated with chaotropic agents and trypsin; the fraction also displayed a tendency to aggregate in the absence of high salt. This property and the affinity of proteins in this fraction for sulfated polysaccharides indicate that specific hydrophilic interactions may play a significant role in sperm-zona attachments.     

Con A诱导植物原生质体凝集作用的研究

不同种的植物表现出有不同的凝集反应。随着温度及Con A浓度的提高,凝集作用增强,并且低温(5～6℃)对裸大麦叶原生质体的凝集作用的影响随温度恢复而可以消失。处于无生长素条件下而脱分化的烟瘤、生长素诱导脱分化的烟草愈伤组织以及处于正常分化状态的叶,三者原生质体间的凝集反应有明显差异。而植物激素IAA、6-BA及GA_3对凝集作用的影响表明,GA_3能引起凝集效应减弱。     

Acute inhibition of PMCA4, but not global ablation,reduces blood pressure and arterial contractility via a nNOS‐dependent mechanism

Cardiovascular disease is the world's leading cause of morbidity and mortality, with high blood pressure (BP) contributing to increased severity and number of adverse outcomes. Plasma membrane calcium ATPase 4 (PMCA4) has been previously shown to modulate systemic BP. However, published data are conflicting, with both overexpression and inhibition of PMCA4 in vivo shown to increase arterial contractility. Hence, our objective was to determine the role of PMCA4 in the regulation of BP and to further understand how PMCA4 functionally regulates BP using a novel specific inhibitor to PMCA4, aurintricarboxylic acid (ATA). Our approach assessed conscious BP and contractility of resistance arteries from PMCA4 global knockout (PMCA4KO) mice compared to wild‐type animals. Global ablation of PMCA4 had no significant effect on BP, arterial structure or isolated arterial contractility. ATA treatment significantly reduced BP and arterial contractility in wild‐type mice but had no significant effect in PMCA4KO mice. The effect of ATAin vivo and ex vivo was abolished by the neuronal nitric oxide synthase (nNOS) inhibitor Vinyl‐l ‐NIO. Thus, this highlights differences in the effects of PMCA4 ablation and acute inhibition on the vasculature. Importantly, for doses here used, we show the vascular effects of ATA to be specific for PMCA4 and that ATA may be a further experimental tool for elucidating the role of PMCA4.     

Extracellular vesicles have variable dose‐dependent effects on cultured draining cells in the eye

The role of extracellular vesicles (EVs) as signal mediators has been described in many biological fields. How many EVs are needed to deliver the desired physiological signal is yet unclear. Using a normal trabecular meshwork (NTM) cell culture exposed to non‐pigmented ciliary epithelium (NPCE)–derived EVs, a relevant model for studying the human ocular drainage system, we addressed the EVs dose–response effects on the Wnt signaling. The objective of the study was to investigate the dosing effects of NPCE‐derived EVs on TM Wnt signaling. EVs were isolated by PEG 8000 method from NPCE and RPE cells (used as controls) conditioned media. Concentrations were determined by Tunable Resistive Pulse Sensing method. Various exosomes concentration were incubated with TM cells, for the determination of mRNA (β‐Catenin, Axin2 and LEF1) and protein (β‐Catenin, GSK‐3β) expression using real‐time quantitative PCR and Western blot, respectively. Exposure of NTM cells for 8 hrs to low EVs concentrations was associated with a significant decreased expression of β‐Catenin, GSK‐3β, as opposed to exposure to high exosomal concentrations. Pro‐MMP9 and MMP9 activities were significantly enhanced in NTM cells treated with high EV concentrations of (X10) as compared to low EV concentrations of either NPCE‐ or RPE‐derived EVs and to untreated control. Our data support the concept that EVs biological effects are concentration‐dependent at their target site. Specifically in the present study, we described a general dose–response at the gene and MMPs activity and a different dose–response regarding key canonical Wnt proteins expression.     

Cargo crowding at actin‐rich regions along axons causes local traffic jams

Steady axonal cargo flow is central to the functioning of healthy neurons. However, a substantial fraction of cargo in axons remains stationary up to several minutes. We examine the transport of precursors of synaptic vesicles (pre‐SVs), endosomes and mitochondria in Caenorhabditis elegans touch receptor neurons, showing that stationary cargo are predominantly present at actin‐rich regions along the neuronal process. Stationary vesicles at actin‐rich regions increase the propensity of moving vesicles to stall at the same location, resulting in traffic jams arising from physical crowding. Such local traffic jams at actin‐rich regions are likely to be a general feature of axonal transport since they also occur in Drosophila neurons. Repeated touch stimulation of C. elegans reduces the density of stationary pre‐SVs, indicating that these traffic jams can act as both sources and sinks of vesicles. This suggests that vesicles trapped in actin‐rich regions are functional reservoirs that may contribute to maintaining robust cargo flow in the neuron. A video abstract of this article can be found at: Video S1 ; Video S2      

Cell sheets in cell therapies

This review aims to provide a broad introduction to the use of cell sheets and the role of materials in the delivery of cell sheets to patients within a clinical setting. Traditionally, cells sheets have been, and currently are, fabricated using established and accepted cell culture methods within standard formats (e.g., petri dishes) utilizing biological substrates. Synthetic surfaces provide a far more versatile system for culturing and delivering cell sheets. This has the potential to positively affect quality, and efficient, localized cell delivery has a significant impact on patient outcome and on the overall cost of goods. We highlight current applications of these advanced carriers and future applications of these surfaces and cell sheets with an emphasis both on clinical use and regulatory requirements.     

Extracellular vesicles released from mesenchymal stromal cells stimulate bone growth in osteogenesis imperfecta

Background

Systemic infusion of mesenchymal stromal cells (MSCs) has been shown to induce acute acceleration of growth velocity in children with osteogenesis imperfecta (OI) despite minimal engraftment of infused MSCs in bones. Using an animal model of OI we have previously shown that MSC infusion stimulates chondrocyte proliferation in the growth plate and that this enhanced proliferation is also observed with infusion of MSC conditioned medium in lieu of MSCs, suggesting that bone growth is due to trophic effects of MSCs. Here we sought to identify the trophic factor secreted by MSCs that mediates this therapeutic activity.