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Per Ekström – One of the best experts on this subject based on the ideXlab platform.

  • Retrograde axonal transport of locally synthesized proteins, e.g., actin and heat shock protein 70, in regenerating Adult Frog sciatic sensory axons.
    Journal of neuroscience research, 1994
    Co-Authors: Magnus Edbladh, Per Ekström, Anders Edström
    Abstract:

    The local synthesis and subsequent retrograde axonal transport of [35S]methionine-labelled proteins was studied in the in vitro regenerating Adult Frog sciatic sensory axons. By the use of a three compartment culture system, proteins in the outgrowth region were selectively labelled. After 2 days in culture a rise in TCA-insoluble radioactivity was detected in the dorsal root ganglia, which could be prevented by the addition of vinblastine or 2,4-dinitrophenol to the nerve proximal to the crush site. Two-dimensional polyacrylamide gel electrophoresis of ganglionic proteins revealed a pattern of 35 labelled polypeptides with apparent molecular masses (Mm) ranging from < 15 to 95 kDa and with isoelectric points (pI) ranging from 4.5 to 6.5. The major ones, representing about 75% of the activity in a gel, were of Mm/pI 47/5.4, 48/6.1,. 57/6.0, 62/5.2, 65/4.9-5.0, 65/5.2, and 81/5.4 respectively. One of these polypeptides (47/5.4) was identified as actin and another (81/5.4) as a member of the heat shock protein 70 family. The spots at 65/4.9-5.0 were tubulin isoforms. There was a striking similarity between transported proteins on one hand, and proteins synthesized in the injured nerve on the other, with respect to the Mm/pI of at least 14 protein species. The results suggest that a selected set of proteins, synthesized by non-neuronal cells, e.g., Schwann cells, is transferred to the ganglionic cell bodies by retrograde axonal transport.

  • Adenosine inhibition of the regeneration in vitro of Adult Frog sciatic sensory axons.
    Brain research, 1992
    Co-Authors: Anders Edström, Magnus Edbladh, Per Ekström
    Abstract:

    The sensory axons of the Adult Frog sciatic nerve have earlier been shown to regenerate in vitro. If a local test crush is made at the initiation of culturing, regeneration starts after 3.4 days and proceeds at a rate of about 0.8-0.9 mm/day for several days. In the present experiments regeneration was inhibited by adenosine in a reversible and dose-dependent fashion. Similarly, both an adenosine analogue, 2-chloroadenosine (2-CA), and a non-hydrolyzable ATP analogue, AMP-PNP, reduced the outgrowth of sensory axons. The effect of adenosine was partially antagonized by theophylline at a critical concentration. Using a compartmental system, it could clearly be shown that adenosine exerted its effects at the outgrowth region. Adenosine, 2-CA, and AMP-PNP were also found to inhibit the proliferation of Schwann cells in the regenerating nerve. Various experiments showed that the latter can not explain the outgrowth inhibitory effects, which could be mediated by adenosine receptors associated with the elongating axons.

  • Effects of protein kinase inhibitors on regeneration in vitro of Adult Frog sciatic sensory axons.
    Journal of neuroscience research, 1992
    Co-Authors: Per Ekström, H. Bergstrand, Anders Edström
    Abstract:

    The effects of protein kinase inhibitors on regeneration in vitro of Adult Frog sciatic sensory axons were tested. Regeneration of crush‐injured nerves for 8 days in serum‐free medium was inhibited by staurosporine (100 nM) and H‐7 (100 μM), which are both known to inhibit protein kinase C. With the use of a compartmented culture system it could be shown that H‐7 exerted both local (outgrowth region) and central (ganglia) effects, the latter being more pronounced. The local effects could be due to reduction of Schwann cell proliferation by H‐7. Immunohisto‐chemistry demonstrated the presence of protein kinase C in neuronal cell bodies but not in axonal processes. Proliferation of Schwann cells was accompanied by increased protein kinase C immunoreactivity at the site of injury. H‐7 caused a selective inhibition in the incorporation of radioactive phosphate into one 74 kDa protein of both ganglia and nerve but also a more general decrease in protein labelling. The results show that protein phosphorylations, possibly mediated by protein kinase C, are involved in regeneration‐related mechanisms operating at both local and central levels in the Adult Frog sciatic sensory axons. (Less)

Anders Edström – One of the best experts on this subject based on the ideXlab platform.

  • Retrograde axonal transport of locally synthesized proteins, e.g., actin and heat shock protein 70, in regenerating Adult Frog sciatic sensory axons.
    Journal of neuroscience research, 1994
    Co-Authors: Magnus Edbladh, Per Ekström, Anders Edström
    Abstract:

    The local synthesis and subsequent retrograde axonal transport of [35S]methionine-labelled proteins was studied in the in vitro regenerating Adult Frog sciatic sensory axons. By the use of a three compartment culture system, proteins in the outgrowth region were selectively labelled. After 2 days in culture a rise in TCA-insoluble radioactivity was detected in the dorsal root ganglia, which could be prevented by the addition of vinblastine or 2,4-dinitrophenol to the nerve proximal to the crush site. Two-dimensional polyacrylamide gel electrophoresis of ganglionic proteins revealed a pattern of 35 labelled polypeptides with apparent molecular masses (Mm) ranging from < 15 to 95 kDa and with isoelectric points (pI) ranging from 4.5 to 6.5. The major ones, representing about 75% of the activity in a gel, were of Mm/pI 47/5.4, 48/6.1,. 57/6.0, 62/5.2, 65/4.9-5.0, 65/5.2, and 81/5.4 respectively. One of these polypeptides (47/5.4) was identified as actin and another (81/5.4) as a member of the heat shock protein 70 family. The spots at 65/4.9-5.0 were tubulin isoforms. There was a striking similarity between transported proteins on one hand, and proteins synthesized in the injured nerve on the other, with respect to the Mm/pI of at least 14 protein species. The results suggest that a selected set of proteins, synthesized by non-neuronal cells, e.g., Schwann cells, is transferred to the ganglionic cell bodies by retrograde axonal transport.

  • Adenosine inhibition of the regeneration in vitro of Adult Frog sciatic sensory axons.
    Brain research, 1992
    Co-Authors: Anders Edström, Magnus Edbladh, Per Ekström
    Abstract:

    The sensory axons of the Adult Frog sciatic nerve have earlier been shown to regenerate in vitro. If a local test crush is made at the initiation of culturing, regeneration starts after 3.4 days and proceeds at a rate of about 0.8-0.9 mm/day for several days. In the present experiments regeneration was inhibited by adenosine in a reversible and dose-dependent fashion. Similarly, both an adenosine analogue, 2-chloroadenosine (2-CA), and a non-hydrolyzable ATP analogue, AMP-PNP, reduced the outgrowth of sensory axons. The effect of adenosine was partially antagonized by theophylline at a critical concentration. Using a compartmental system, it could clearly be shown that adenosine exerted its effects at the outgrowth region. Adenosine, 2-CA, and AMP-PNP were also found to inhibit the proliferation of Schwann cells in the regenerating nerve. Various experiments showed that the latter can not explain the outgrowth inhibitory effects, which could be mediated by adenosine receptors associated with the elongating axons.

  • Effects of protein kinase inhibitors on regeneration in vitro of Adult Frog sciatic sensory axons.
    Journal of neuroscience research, 1992
    Co-Authors: Per Ekström, H. Bergstrand, Anders Edström
    Abstract:

    The effects of protein kinase inhibitors on regeneration in vitro of Adult Frog sciatic sensory axons were tested. Regeneration of crush‐injured nerves for 8 days in serum‐free medium was inhibited by staurosporine (100 nM) and H‐7 (100 μM), which are both known to inhibit protein kinase C. With the use of a compartmented culture system it could be shown that H‐7 exerted both local (outgrowth region) and central (ganglia) effects, the latter being more pronounced. The local effects could be due to reduction of Schwann cell proliferation by H‐7. Immunohisto‐chemistry demonstrated the presence of protein kinase C in neuronal cell bodies but not in axonal processes. Proliferation of Schwann cells was accompanied by increased protein kinase C immunoreactivity at the site of injury. H‐7 caused a selective inhibition in the incorporation of radioactive phosphate into one 74 kDa protein of both ganglia and nerve but also a more general decrease in protein labelling. The results show that protein phosphorylations, possibly mediated by protein kinase C, are involved in regeneration‐related mechanisms operating at both local and central levels in the Adult Frog sciatic sensory axons. (Less)

Leon Grayfer – One of the best experts on this subject based on the ideXlab platform.

  • Amphibian (Xenopus laevis) Interleukin-8 (CXCL8): A Perspective on the Evolutionary Divergence of Granulocyte Chemotaxis.
    Frontiers in immunology, 2018
    Co-Authors: Daphne V. Koubourli, Amulya Yaparla, Milan Popovic, Leon Grayfer
    Abstract:

    The glutamic acid-leucine-arginine (ELR) motif is a hallmark feature shared by mammalian inflammatory CXC chemokines such the granulocyte chemo-attractant CXCL8 (interleukin-8, IL-8). By contrast, most teleost fish inflammatory chemokines lack this motif. Interestingly, the amphibian Xenopus laevis encodes multiple isoforms of CXCL8, one of which (CXCL8a) possesses an ELR motif, while another (CXCL8b) does not. These CXCL8 isoforms exhibit distinct expression patterns during Frog development and following immune challenge of animals and primary myeloid cultures. To define potential functional differences between these X. laevis CXCL8 chemokines, we produced them in recombinant form (rCXCL8a and rCXCL8b) and performed dose-response chemotaxis assays. Our results indicate that compared to rCXCL8b, rCXCL8a is a significantly more potent chemo-attractant of in vivo-derived tadpole granulocytes and of in vitro-differentiated Frog bone marrow granulocytes. The mammalian CXCL8 mediates its effects through two distinct chemokine receptors, CXCR1 and CXCR2 and our pharmacological inhibition of these receptors in Frog granulocytes indicates that the X. laevis CXCL8a and CXCL8b both chemoattract tadpole and Adult Frog granulocytes by engaging CXCR1 and CXCR2. To delineate which Frog cells are recruited by CXCL8a and CXCL8b in vivo, we injected tadpoles and Adult Frogs intraperitoneally with rCXCL8a or rCXCL8b and recovered the accumulated cells by lavage. Our transcriptional and cytological analyses of these tadpole and Adult Frog peritoneal exudates indicate that they are comprised predominantly of granulocytes. Interestingly, the granulocytes recruited into the tadpole, but not Adult Frog peritonea by rCXCL8b, express significantly greater levels of several pan immunosuppressive genes.

  • Elicitation of Xenopus laevis Tadpole and Adult Frog Peritoneal Leukocytes.
    Cold Spring Harbor protocols, 2018
    Co-Authors: Leon Grayfer
    Abstract:

    Peritoneal lavage of Xenopus laevis tadpoles and Adult Frogs is a reliable way of isolating resident and/or recruited innate immune populations. This protocol details the isolation of tadpole and Adult amphibian (Xenopus laevis) peritoneal leukocytes. The isolated cells are comprised predominantly of innate immune populations and chiefly of mononuclear and polymorphonuclear granulocytes. As described here, these cells are typically elicited by peritoneal injections of animals with heat-killed Escherichia coli, causing peritoneal accumulation of inflammatory cell populations, which are then isolated from the stimulated animals by lavage. E. coli-mediated elicitation of tadpole and Adult peritoneal leukocytes greatly enhances the total numbers of recovered cells, at the cost of their inflammatory activation. Conversely, lavage may be performed on naive, unstimulated animals to isolate nonactivated cells with much lower yield. This protocol represents a reliable means of deriving tadpole and Adult Frog innate immune cell populations, and the conditions of the stimulation may be amended to suit the specifics of a given experimental design.

  • Image_1_Amphibian (Xenopus laevis) Interleukin-8 (CXCL8): A Perspective on the Evolutionary Divergence of Granulocyte Chemotaxis.TIF
    , 2018
    Co-Authors: Daphne V. Koubourli, Amulya Yaparla, Milan Popovic, Leon Grayfer
    Abstract:

    The glutamic acid-leucine-arginine (ELR) motif is a hallmark feature shared by mammalian inflammatory CXC chemokines such the granulocyte chemo-attractant CXCL8 (interleukin-8, IL-8). By contrast, most teleost fish inflammatory chemokines lack this motif. Interestingly, the amphibian Xenopus laevis encodes multiple isoforms of CXCL8, one of which (CXCL8a) possesses an ELR motif, while another (CXCL8b) does not. These CXCL8 isoforms exhibit distinct expression patterns during Frog development and following immune challenge of animals and primary myeloid cultures. To define potential functional differences between these X. laevis CXCL8 chemokines, we produced them in recombinant form (rCXCL8a and rCXCL8b) and performed dose-response chemotaxis assays. Our results indicate that compared to rCXCL8b, rCXCL8a is a significantly more potent chemo-attractant of in vivo-derived tadpole granulocytes and of in vitro-differentiated Frog bone marrow granulocytes. The mammalian CXCL8 mediates its effects through two distinct chemokine receptors, CXCR1 and CXCR2 and our pharmacological inhibition of these receptors in Frog granulocytes indicates that the X. laevis CXCL8a and CXCL8b both chemoattract tadpole and Adult Frog granulocytes by engaging CXCR1 and CXCR2. To delineate which Frog cells are recruited by CXCL8a and CXCL8b in vivo, we injected tadpoles and Adult Frogs intraperitoneally with rCXCL8a or rCXCL8b and recovered the accumulated cells by lavage. Our transcriptional and cytological analyses of these tadpole and Adult Frog peritoneal exudates indicate that they are comprised predominantly of granulocytes. Interestingly, the granulocytes recruited into the tadpole, but not Adult Frog peritonea by rCXCL8b, express significantly greater levels of several pan immunosuppressive genes.

Frank Scalia – One of the best experts on this subject based on the ideXlab platform.

  • persistence of graded epha ephrin a expression in the Adult Frog visual system
    The Journal of Comparative Neurology, 2003
    Co-Authors: Helene Bach, David A Feldheim, John G Flanagan, Frank Scalia
    Abstract:

    Many studies have demonstrated the involvement of the EphA family of receptor tyrosine kinases and their ligands, ephrin-A2 and -A5, in the development of the temporonasal axis of the retinotectal/collicular map, but the role of these molecules in optic nerve regeregeneration has not been well studied. Noting that the characteristic gradients of the EphA/ephrin-A family that are expressed topographically in the retina and tectum of embryonic chicks and mice tend to disappear after birth, we took as our starting point an analysis of EphA and ephrin-A expression in leopard Frogs (Rana pipiens and utricularia), species capable of regenerating the retinotectal map as Adults. For the EphA family to be involved in the regeneration, one would expect these topographic gradients to persist in the Adult or, if downregulated after metamorphosis, to be reexpressed after optic nervnerve injury. Using EphA3 receptor and ephrin-A5 ligand alkaline phosphatase in situ affinity probes (RAP and LAP, respectively) in whole-mount applications, we report that reciprocally complementary gradients of RAP and LAP binding persist in the optic tract and optic tectum of postmetamorphic Frogs, including mature Adults. EphA expression in temporal retinal axons in the optic tract was significantly reduced after nerve section but returned during regeneration. However, ephrin-A expression in the tectal parenchyma was not significantly elevated by either eye removal, with degeneration of optic axons, or during regeneration of the retinotectal projection. Thus, the present study has demonstrated a persisting expression of EphA/ephrin-A family members in the retinal axons and tectal parenchyma that may help guide regenerating fibers, but we can offer no evidence for an upregulation of ephrin-A expression in conjunction with optic nervnerve injury. J. Comp. Neurol. 467:549–565, 2003. © 2003 Wiley-Liss, Inc.

  • Quantitative study of the tectally projecting retinal ganglion cells in the Adult Frog. II. Cell survival and functional recovery after optic nerve transection
    The Journal of comparative neurology, 1991
    Co-Authors: Eric L. Singman, Frank Scalia
    Abstract:

    It is known from previous work that ganglion cells disappear from the retina in significant numbers during optic nerve regeregeneration in the Adult Frog, In the present study, the population sine of surviving ganglion cells that have returned axon terminals to the correct tectal loci was estimated by counts of retrogradely labeled cells in retina-flat-mounts after tectal injections of HRP. Bilaterally symmetric injections were delivered to allow comparison of the normal and affected retinas. The Frogs studied had regenerated the left optic nerve and had visually guided behaviors initiated by the recovered eye (see below). The proportion of tectally projecting ganglion cells in the normal retinas and in retinas of normal Frogs studied in parallel ranged from 83–86% (Singman and Scalia: J. Comp. Neurol. 302: 792–809, 1991), In the affected retinas, the subpopulation of tectally projecting cells was reduced by 40–90% after regeneration, and the relative size of this subpopulation ranged from 67–86%. The optic tectum was injected unilaterally in one specimen, on the side ipsilateral to the regenerated (left) optic nerve. The HRP-labeled ganglion cells in the ipsilateral (left) retina accounted for only 0.8% of the surviving ganglion cells in this animal, whereas it was previously found that the ipsilateral tectally projecting ganglion cells normally amount to 0.9–2.3% (Singman and Scalia, op. cit.) In Frogs recovering from transection of the left optic nerve, the frequency, latency, and accuracy of the prey-acquisition responses initiated by the recovering eye were compared with those initiated by the normal eye. Mealworms or lure dummies were used to stimulate prey acquisition. In one experiment, the stimuli were presented unilaterally in the monocular fields of Frogs permitted to use both eyes. Prior to the fourteenth postoperative week, the affected eye initiated responses of abnormally long latency and low frequency. In contrast, responses initiated by the affected eye after 14 weeks appeared to be normal in all respects. In another experiment, the normal eye was sutured shut in some Frogs recovering for at least 24 weeks and then the affected eye was retested. The affected eye was capable of consistently initiating brisk and accurate prey acquisition. In a final experiment, two stimuli were presented simultaneously in bilaterally symmetric regions of the monocular fields of Frogs surviving at least 42 weeks. These fully recovered Frogs showed no preference for using either the normal or the recovered eye. Despite severe loss of tectally projecting ganglion cells during optic nerve regeregeneration, Frogs are capable of apparently normal visual responses in prey acquisition tests.

Magnus Edbladh – One of the best experts on this subject based on the ideXlab platform.

  • Retrograde axonal transport of locally synthesized proteins, e.g., actin and heat shock protein 70, in regenerating Adult Frog sciatic sensory axons.
    Journal of neuroscience research, 1994
    Co-Authors: Magnus Edbladh, Per Ekström, Anders Edström
    Abstract:

    The local synthesis and subsequent retrograde axonal transport of [35S]methionine-labelled proteins was studied in the in vitro regenerating Adult Frog sciatic sensory axons. By the use of a three compartment culture system, proteins in the outgrowth region were selectively labelled. After 2 days in culture a rise in TCA-insoluble radioactivity was detected in the dorsal root ganglia, which could be prevented by the addition of vinblastine or 2,4-dinitrophenol to the nerve proximal to the crush site. Two-dimensional polyacrylamide gel electrophoresis of ganglionic proteins revealed a pattern of 35 labelled polypeptides with apparent molecular masses (Mm) ranging from < 15 to 95 kDa and with isoelectric points (pI) ranging from 4.5 to 6.5. The major ones, representing about 75% of the activity in a gel, were of Mm/pI 47/5.4, 48/6.1,. 57/6.0, 62/5.2, 65/4.9-5.0, 65/5.2, and 81/5.4 respectively. One of these polypeptides (47/5.4) was identified as actin and another (81/5.4) as a member of the heat shock protein 70 family. The spots at 65/4.9-5.0 were tubulin isoforms. There was a striking similarity between transported proteins on one hand, and proteins synthesized in the injured nerve on the other, with respect to the Mm/pI of at least 14 protein species. The results suggest that a selected set of proteins, synthesized by non-neuronal cells, e.g., Schwann cells, is transferred to the ganglionic cell bodies by retrograde axonal transport.

  • Adenosine inhibition of the regeneration in vitro of Adult Frog sciatic sensory axons.
    Brain research, 1992
    Co-Authors: Anders Edström, Magnus Edbladh, Per Ekström
    Abstract:

    The sensory axons of the Adult Frog sciatic nerve have earlier been shown to regenerate in vitro. If a local test crush is made at the initiation of culturing, regeneration starts after 3.4 days and proceeds at a rate of about 0.8-0.9 mm/day for several days. In the present experiments regeneration was inhibited by adenosine in a reversible and dose-dependent fashion. Similarly, both an adenosine analogue, 2-chloroadenosine (2-CA), and a non-hydrolyzable ATP analogue, AMP-PNP, reduced the outgrowth of sensory axons. The effect of adenosine was partially antagonized by theophylline at a critical concentration. Using a compartmental system, it could clearly be shown that adenosine exerted its effects at the outgrowth region. Adenosine, 2-CA, and AMP-PNP were also found to inhibit the proliferation of Schwann cells in the regenerating nerve. Various experiments showed that the latter can not explain the outgrowth inhibitory effects, which could be mediated by adenosine receptors associated with the elongating axons.