Enhanced production of mouse hybridomas to picomoles of antigen using EL-4 conditioned media with an in vitro immunization protocol

Summary We report here that the use of murine thymoma cell EL-4 conditioned medium enhances hybridoma yield in a low-antigen dose in vitro immunization protocol. This improved protocol allowed the production of a panel of monoclonal antibodies toDrosophila yolk proteins using less than 1 nanomole of antigen. We believe this refinement will be valuable for the application of hybridoma technology to biologically active materials that are hard to isolate and purify due to their low concentration in the biological fluids. This research was supported by the College of Agriculture and Life Sciences, University of Maryland, USDA-University of Maryland Cooperative Editor's Statement This observation should simplify in vitro immunization approaches and shed new light on the factors required for the in vitro immune response. Wallace L. McKeehan     

Satellite Glial Cells and Astrocytes,a Comparative Review

Astroglia are neural cells, heterogeneous in form and function, which act as supportive elements of the central nervous system; astrocytes contribute to all aspects of neural functions in health and disease. Through their highly ramified processes, astrocytes form close physical contacts with synapses and blood vessels, and are integrated into functional syncytia by gap junctions. Astrocytes interact among themselves and with other cells types (e.g., neurons, microglia, blood vessel cells) by an elaborate repertoire of chemical messengers and receptors; astrocytes also influence neural plasticity and synaptic transmission through maintaining homeostasis of neurotransmitters, K⁺ buffering, synaptic isolation and control over synaptogenesis and synaptic elimination. Satellite glial cells (SGCs) are the most abundant glial cells in sensory ganglia, and are believed to play major roles in sensory functions, but so far research into SGCs attracted relatively little attention. In this review we compare SGCs to astrocytes with the purpose of using the vast knowledge on astrocytes to explore new aspects of SGCs. We survey the main properties of these two cells types and highlight similarities and differences between them. We conclude that despite the much greater diversity in morphology and signaling mechanisms of astrocytes, there are some parallels between them and SGCs. Both types serve as boundary cells, separating different compartments in the nervous system, but much more needs to be learned on this aspect of SGCs. Astrocytes and SGCs employ chemical messengers and calcium waves for intercellular signaling, but their significance is still poorly understood for both cell types. Both types undergo major changes under pathological conditions, which have a protective function, but an also contribute to disease, and chronic pain in particular. The knowledge obtained on astrocytes is likely to benefit future research on SGCs.

     

Ceruloplasmin and myeloperoxidase in complex affect the enzymatic properties of each other

Ceruloplasmin (CP), the multicopper oxidase of plasma, interacts with myeloperoxidase (MPO), an enzyme of leukocytes, and inhibits its peroxidase and chlorinating activity. Studies on the enzymatic properties shows that CP behaves as a competitive inhibitor impeding the binding of aromatic substrates to the active centre of MPO. The contact between CP and MPO probably entails conformational changes close to the p-phenylenediamine binding site in CP, which explains the observed activation by MPO of the substrate's oxidation. CP subjected to partial proteolysis was virtually unable to inhibit activity of MPO. The possible protein–protein interface is comprised of the area near active site of MPO and the loop linking domains 5 and 6 in CP. One of the outcomes of this study is the finding of a new link between antioxidant properties of CP and its susceptibility to proteolysis.     

Differentiation of adipose-derived stem cells into Schwann cell phenotype induces expression of P2X receptors that control cell death

Schwann cells (SCs) are fundamental for development, myelination and regeneration in the peripheral nervous system. Slow growth rate and difficulties in harvesting limit SC applications in regenerative medicine. Several molecules, including receptors for neurosteroids and neurotransmitters, have been suggested to be implicated in regulating physiology and regenerative potential of SCs. Adipose-derived stem cells (ASCs) can be differentiated into SC-like phenotype (dASC) sharing morphological and functional properties with SC, thus representing a valid SC alternative. We have previously shown that dASC express γ-aminobutyric-acid receptors, which modulate their proliferation and neurotrophic potential, although little is known about the role of other neurotransmitters in ASC. In this study, we investigated the expression of purinergic receptors in dASC. Using reverse transriptase (RT)-PCR, western blot analyses and immunocytochemistry, we have demonstrated that ASCs express P2X₃, P2X₄ and P2X₇ purinoceptors. Differentiation of ASCs towards glial phenotype was accompanied by upregulation of P2X₄ and P2X₇ receptors. Using Ca²⁺-imaging techniques, we have shown that stimulation of purinoceptors with adenosine 5′-triphosphate (ATP) triggers intracellular Ca²⁺ signals, indicating functional activity of these receptors. Whole-cell voltage clamp recordings showed that ATP and BzATP induced ion currents that can be fully inhibited with specific P2X₇ antagonists. Finally, using cytotoxicity assays we have shown that the increase of intracellular Ca²⁺ leads to dASC death, an effect that can be prevented using a specific P2X₇ antagonist. Altogether, these results show, for the first time, the presence of functional P2X₇ receptors in dASC and their link with critical physiological processes such as cell death and survival. The presence of these novel pharmacological targets in dASC might open new opportunities for the management of cell survival and neurotrophic potential in tissue engineering approaches using dASC for nerve repair.     

Alterations in the Function of Endoplasmic Reticulum and Neuronal Signalling

For many years, the endoplasmic reticulum (ER) was considered to be involved in rapid signalling events due to its ability to serve as a dynamic calcium store capable of accumulating large amounts of Ca²⁺ ions and of releasing them in response to physiological stimulation. Recent data significantly increased the importance of the ER as a signalling organelle, by demonstrating that the ER is associated with specific pathways regulating long-lasting adaptive processes and controlling cell survival. The ER lumen is enriched by enzymatic systems involved in protein synthesis and correcting post-translational folding of these proteins. The processes of post-translational protein processing are controlled by a class of specific enzymes known as chaperones, which in turn are regulated by the free Ca²⁺ concentration within the ER lumen ([Ca²⁺]_L). At the same time, a high [Ca²⁺]_L determines the ability of the ER to generate cytosolic Ca²⁺ signals. Thus, the ER is able to produce signals interacting within different temporal domains. Fast ER signals result from Ca²⁺ release via specific Ca²⁺-release channels and from rapid movements of Ca²⁺ ions within the ER lumen (calcium tunneling). Long-lasting signals involve Ca²⁺-dependent regulation of chaperones with subsequent changes in protein processing and synthesis. Any malfunctions in the ER Ca²⁺ homeostasis result in accumulation of unfolded proteins, which in turn activates several signalling systems aimed at appropriate compensatory responses or (in the case of severe ER dysregulation) in cellular pathology and death (ER stress responses). Thus, the Ca²⁺ ion emerges as a messenger molecule, which integrates various signals within the ER: fluctuations of the [Ca²⁺]_L induced by signals originating at the level of the plasmalemma (i.e., Ca²⁺ entry or activation of the metabotropic receptors) regulate in turn protein synthesis and processing via generating secondary signalling events between the ER and the nucleus.     

Endoplasmic reticulum Ca(2+) homeostasis and neuronal death

The endoplasmic reticulum (ER) is a universal signalling organelle, which regulates a wide range of neuronal functional responses. Calcium release from the ER underlies various forms of intracellular Ca²⁺ signalling by either amplifying Ca²⁺ entry through voltage-gated Ca²⁺ channels by Ca²⁺-induced Ca²⁺ release (CICR) or by producing local or global cytosolic calcium fluctuations following stimulation of metabotropic receptors through inositol-1,4,5-trisphosphate-induced Ca²⁺ release (IICR). The ER Ca²⁺ store emerges as a single interconnected pool, thus allowing for a long-range Ca²⁺ signalling via intra-ER tunnels. The fluctuations of intra-ER free Ca²⁺ concentration regulate the activity of numerous ER resident proteins responsible for post-translational protein folding and modification. Disruption of ER Ca²⁺ homeostasis results in the developing of ER stress response, which in turn controls neuronal survival. Altered ER Ca²⁺ handling may be involved in pathogenesis of various, neurodegenerative diseases including brain ischemia and Alzheimer dementia.     

Functional specialization of presynaptic Cav2.3 Ca2+ channels

Ca2+ influx into presynaptic terminals via voltage-dependent Ca2+ channels triggers fast neurotransmitter release as well as different forms of synaptic plasticity. Using electrophysiological and genetic techniques we demonstrate that presynaptic Ca2+ entry through Cav2.3 subunits contributes to the induction of mossy fiber LTP and posttetanic potentiation by brief trains of presynaptic action potentials while they do not play a role in fast synaptic transmission, paired-pulse facilitation, or frequency facilitation. This functional specialization is most likely achieved by a localization remote from the release machinery and by a Cav2.3 channel-dependent facilitation of presynaptic Ca2+ influx. Thus, the presence of Cav2.3 channels boosts the accumulation of presynaptic Ca2+ triggering presynaptic LTP and posttetanic potentiation without affecting the low release probability that is a prerequisite for the enormous plasticity displayed by mossy fiber synapses.