CXCL12/SDF-1-Dependent Retinal Migration of Endogenous Bone Marrow-Derived Stem Cells Improves Visual Function after Pharmacologically Induced Retinal Degeneration.

Mobilized bone marrow-derived stem cells (BMSC) have been discussed as an alternative strategy for endogenous repair. Thereby, different approaches for BMSC mobilization have been pursued. Herein, the role of a newly discovered oligonucleotide for retinal homing and regeneration capability of BMSCs was investigated in the sodium iodate (NaIO3) model of retinal degeneration. Mobilization was achieved in GFP-chimera with NOX-A12, a CXC-motif chemokine ligand 12 (CXCL12)/stromal cell-derived factor 1 (SDF-1)-neutralizing L-aptamer. BMSC homing was directed by intravitreal SDF-1 injection. Visual acuity was measured using the optokinetic reflex. Paraffin cross sections were stained with hematoxylin and eosin for retinal thickness measurements. Immunohistochemistry was performed to investigate the expression of cell-specific markers after mobilization. A single dose of NOX-A12 induced significant mobilization of GFP+ cells which were found in all layers within the degenerating retina. An additional intravitreal injection of SDF-1 increased migration towards the site of injury. Thereby, the number of BMSCs (Sca-1+) found in the damaged retina increased whereas a decrease of activated microglia (Iba-1+) was found. The mobilization led to significantly increased visual acuity. However, no significant changes in retinal thickness or differentiation towards retinal cell types were detected. Systemic mobilization by a single dose of NOX-A12 showed increased homing of BMSCs into the degenerated retina, which was associated with improved visual function when injection of SDF-1 was additionally performed. The redistribution of the cells to the site of injury combined with their observed beneficial effects support the endogenous therapeutic strategy for retinal repair.

Cytotoxicity evaluation of polymer-derived ceramics for pacemaker electrode applications.

Ceramics are known to be chemically stable, and the possibility to electrically dope polymer-derived ceramics makes it a material of interest for implantable electrode applications. We investigated cytotoxic characteristics of four polymer-derived ceramic candidates with either electrically conductive or insulating properties. Cytotoxicity was assessed by culturing C2C12 myoblast cells under two conditions: by exposing them to material extracts and by putting them directly in contact with material samples. Cell spreading was optically evaluated by comparing microscope observations immediately after the materials insertion and after 24 h culturing. Cell viability (MTT) and mortality (LDH) were quantified after 24-h incubation in contact with the materials. Comparison was made with biocompatible positive references (alumina, platinum, biocompatible stainless steel 1.4435), negative references (latex, stainless steel 1.4301) and controls (no material present in the culture wells). We found that the cytotoxic properties of tested ceramics are comparable to established reference materials. These ceramics, which are reported to be very stable, can be microstructured and electrically doped to a wide range of conductivity and are thus excellent candidates for implantable electrode applications including pacemakers.

Evolution of gnathostome prodynorphin and proenkephalin: characterization of a shark proenkephalin and prodynorphin cDNAs.

Analyses of prodynorphin and proenkephalin cDNAs cloned from the central nervous system of the shark, Heterodontus portusjacksoni, provided additional evidence that these two opioid precursor-coding genes were most likely directly derived from a common ancestral gene. The two cDNAs could be aligned by inserting only seven gaps. The prodynorphin cDNA encodes five opioid sequences which could be aligned to opioid positions B through F in the proenkephalin cDNA. The sequence identity within the opioid positions was 59% at the amino acid level. Shark α-neo-endorphin, dynorphin A, and dynorphin B have amino acid motifs in common with shark met-enkephalin-8, and shark proenkephalin opioid positions E and F, respectively, which have not been observed in other gnathostome prodynorphin and proenkephalin precursor sequences. Shark prodynorphin encodes both kappa (α-neo-endorphin, dynorphin A, and dynorphin B) and delta (met-enkephalin and leu-enkephalin) opioid sequences. Mixed function prodynorphin precursors (encoding both enkephalins and dynorphins) are also found in representatives of the teleost fishes, lungfishes, and amphibians. It appears that only mammals evolved a prodynorphin precursor that exclusively encodes kappa opioid agonists (dynorphins).

Rasagiline interferes with neurodegeneration in the Prph2/rds mouse.

PURPOSE: Rasagiline (N-propargyl-1(R)-aminoindan) is a second-generation propargylamine with neuroprotective effects. We used the Prph2/rds mouse to assess the effect of rasagiline on photoreceptor cell death and to examine the possible modulation of different pathways of programmed cell death. METHODS: The animals were orally treated with various doses of rasagiline from Postnatal Day 1 to 56. Methodological approaches consisted of morphometric analyses of the outer nuclear layer thickness and investigation of apoptotic events using TUNEL (TdT-mediated dUTP-biotin nick end labeling) assay, immunohistochemistry, and immunoblot staining. The expression of programmed cell death marker genes involved in photoreceptor degeneration was studied by quantitative real-time polymerase chain reaction. RESULTS: In the Prph2/rds mouse, treatment resulted in a significant dose-dependent neuroprotection at Postnatal Day 56 and a delay in the induction of apoptotic events at Postnatal Day 14. Programmed cell death marker gene expression showed that several mechanisms were involved in photoreceptor degeneration. Furthermore, rasagiline did not only target apoptosis but also other pathways such as autophagy and inflammation. CONCLUSION: This study showed for the first time significant neuroprotective effects of rasagiline in the retina of Prph2/rds mice through caspase-dependent pathways. However, the activation of caspase-independent programmed cell death pathways that are not affected by rasagiline eventually led to retinal degeneration, but in a delayed manner.

Caspase-3-independent photoreceptor degeneration by N-methyl-N-nitrosourea (MNU) induces morphological and functional changes in the mouse retina.

BACKGROUND: Retinal degeneration is followed by significant changes in the structure and function of photoreceptors in humans and several genetic animal models. However, it is not clear whether similar changes occur when the degeneration is induced pharmacologically. Therefore, our aim was to investigate the influence of retinotoxic N-methyl-N-nitrosourea (MNU) on the function, morphology and underlying molecular pathways of programmed cell death. METHODS: C57/BL6 mice were injected with different doses of MNU, and function was determined by analysing optokinetic reflex measurements and cued water maze results at several time points post-injection. Morphometric measurements were also taken from H&E-stained paraffin eye sections. TUNEL-positive cells and caspase-3 and -6 were detected by immunohistochemistry. To assess the molecular changes leading to cell death, qRT-PCR from neurosensory retina mRNA was performed. RESULTS: The application of MNU led to an instant decrease in function and a delayed decrease in the thickness of the retinal outer nuclear layer. These responses were observed in the absence of any structural changes in the retinal pigment epithelium. The degeneration of the photoreceptor cell layer was highest with 60 mg/kg MNU. The assessment of TUNEL-positive cells visualised cell death after treatment, but no detectable caspase-3 activity was observed concomitant with these changes. qRT-PCR revealed the possible involvement of the inflammatory mediator caspase-1 and endoplasmic reticulum stress-mediated apoptosis by caspase-12. CONCLUSION: MNU leads to the dose-dependent degeneration of photoreceptor cells in mice by caspase-3-independent pathways and is, therefore, a suitable model to study retinal degeneration in an animal model.

Evolution of the opioid/ORL-1 receptor gene family.

In gnathostomes the kappa, mu, delta, ORL-1 receptor genes constitute the opioid/ORL-1 receptor gene family. These genes are most likely the result of two (2R) genome duplication events that occurred during the radiation of the chordates. In stilico analysis of the genome of the lamprey, Petromyzon marius, revealed the partial sequences of four genes that may be the result of a lineage specific genome duplication event in the lamprey lineage. The sequencing of cDNAs from the lamprey CNS supports the assumption that these putative lamprey opioid-like receptor genes are expressed by lamprey neurons. Analysis of gnathostome ORL-1 receptor sequences support the hypothesis that the ORL-1 gene has undergone a transition from an opioid receptor that could bind several types of opioid ligands to a receptor in mammals that can only be activated by the FGGF form of the orphanin ligand.

Tracking the evolution of the proenkephalin gene in tetrapods.

In gnathostomes there is remarkable consistency in the organization of the proenkephalin gene. This opioid precursor encodes seven opioid (YGGF) sequences: five pentapeptide sequences, a met-enkephalin-7 sequence and a met-enkephalin-8 sequence. Yet, within vertebrate lineages there can be distinct sets of pentapeptide opioids (YGGFM or YGGFL). In the Sarcopterygii, the sixth opioid position in lungfishes and anuran amphibian proenkephalin genes encodes a met-enkephalin (YGGFM) sequence. However, in mammalian proenkephalin there is a leu-enkephalin (YGGFL) sequence at this position. This study was done to test the hypothesis that the presence of the leu-enkephalin sequence in mammals is a feature common to amniote vertebrates, but not present in anamniote vertebrates. To resolve this issue, proenkephalin cDNAs were cloned from the urodele amphibians, Amphiuma means and Necturus maculosus, and two amniote vertebrates, the turtle, Chrysemys scripta, and the brown snake, Storeria dekayi. As predicted, a met-enkephalin sequence is present at the sixth opioid position in urodele amphibians; whereas, a leu-enkephalin sequence is present at this opioid site in the reptile proenkephalin sequences. These data are consistent with the conclusion that the transition from a met-enkephalin sequence to a leu-enkephalin sequence at the sixth opioid position in tetrapod proenkephalins occurred in the ancestral proto-reptiles. Phylogenetic analyses, using the Maximum Parsimony and Neighbor-Joining algorithms, of the amphibian proenkephalin sequences supported the position that anuran and urodele amphibians are a monophyletic assemblage. The same analysis of reptile-related proenkephalin sequences, including the deduced amino acid sequence of a partially characterized alligator proenkephalin cDNA, could not conclusively resolve the phylogeny of the major reptilian orders.

Trends in the evolution of the prodynorphin gene in teleosts: cloning of eel and tilapia prodynorphin cDNAs.

The detection of the prodynorphin gene in anuran amphibians and lungfishes may indicate that this gene arose as a result of the duplication of the proenkephalin gene early during the divergence of the Sarcopterygii, or that this gene may predate the divergence of the ray-finned fish and the lobe-finned fish. The cloning of prodynorphin-related genes from the pufferfish and zebrafish supports the latter hypothesis. This study analyzes trends in the radiation of the prodynorphin gene in teleosts. Prodynorphin cDNAs were cloned from the brain of the eel Anguilla rostrata and the Nile tilapia, Oreochromis niloticus. These teleost prodynorphin sequences have distinct alpha-neoendorphin, dynorphin A, and dynorphin B sequences, and a novel opioid sequence, YGGFI. The relationship of these teleost prodynorphin sequences to other actinopterygian and sarcopterygian prodynorphin sequences will be discussed.

Analyzing the radiation of the melanocortins in amphibians: cloning of POMC cDNAs from the pituitary of the urodele amphibians, Amphiuma means and Necturus maculosus.

Proopiomelanocortin (POMC) cDNAs were cloned and sequenced from brain extracts of two species of urodele amphibians: Amphiuma means and Necturus maculosus. Although the two species of urodele amphibians belong to separate families, and do not share a direct common ancestor, the level of primary sequence identity for the open reading of the POMC cDNAs was 90% at the amino acid level and 79% at the nucleotide level. It appears that the POMC gene in these urodele amphibians has been accumulating mutations at the amino acid level at a slower rate than the POMC gene in other sarcopterygian orders.

Trends in the evolution of the proenkephalin and prodynorphin genes in gnathostomes.

The opioid/orphanin gene family provides a model system for analyzing the outcomes of genome duplication events. Recent studies on the proenkephalin gene provide additional evidence that the organizational plan for this gene has been conserved throughout the extensive radiation of the gnathostome vertebrates. However, an analysis of the amino acid sequence of proenkephalin from the zebrafish, Danio rerio, suggests that novel forms of this opioid precursor may be evolving in teleosts. Analyses of sarcopterygian prodynorphin sequences revealed a proenkephalin signature in prodynorphin. Current studies on the opioid/orphanin gene family point to the duplication events that shaped this family occurring prior to the radiation of the gnathostomes.

Trends in the evolution of the proopiomelanocortin gene.

The POMC gene is perhaps the most extensively studied member of the opioid/orphanin gene family. In Phylum Chordata this gene has been characterized in representatives of every class within the Gnathostomata, as well as in one representative agnathan vertebrate, the marine lamprey. This review provides a systematic overview of trends in the evolution of the melanocortins (ACTH/alpha-MSH, beta-MSH, gamma-MSH, and delta-MSH) and beta-endorphin in gnathostomes, and advances the hypothesis that the appearance of gamma-MSH occurred early in the radiation of the gnathostomes. A summary of the extensive work on POMC genes in the marine lamprey is also provided, as well as a reevaluation of the conserved regions in the sequence of CLIP (corticotropin-like-intermediate lobe peptide) in the POMC sequences of the various groups of gnathostomes.

Cloning of prodynorphin cDNAs from the brain of Australian and African lungfish: implications for the evolution of the prodynorphin gene.

In mammals the opioids Met-enkephalin and Leu-enkephalin are derived from a common precursor, proenkephalin, and as a result these neuropeptides are co-localized in enkephalinergic neurons. The mammalian scheme for enkephalinergic networks is not universal for all classes of sarcopterygian vertebrates. In an earlier study, distinct Met- and Leu-enkephalin-positive neurons were detected in the central nervous system (CNS) of the African lungfish, Protopterus annectens. More recently, characterization of proenkephalin cDNAs separately cloned from the CNS of P. annectens and the Australian lungfish, Neoceratodus forsteri, revealed that the proenkephalin gene in these species encodes only Met-enkephalin-related opioids. In the current study a full-length prodynorphin cDNA (accession No. AY 445637) was cloned and sequenced from the CNS of N. forsteri. In addition to encoding alpha-neoendorphin, dynorphin A and dynorphin B sequences unique to the lungfish, two Leu-enkephalin sequences, flanked by paired basic amino acid proteolytic cleavage sites, were detected in this precursor. The partial sequence of a P. annectens prodynorphin cDNA (accession No. AY445638) also encoded a Leu-enkephalin sequence and a novel YGGFF sequence. The presence of the Leu-enkephalin sequence in the lungfish prodynorphin precursors would explain the origin of the distinct Leu-enkephalin-positive neurons found in the African lungfish CNS. The realization that Met-enkephalin and Leu-enkephalin can be derived from distinct opioid-coding precursor genes calls into question the interpretation of comparative immunohistochemical studies that have mapped 'enkephalinergic' networks in non-mammalian vertebrates.

Characterizing a proopiomelanocortin cDNA cloned from the brain of the Bichir, Polypterus senegalus: evaluating phylogenetic relationships among ray-finned fish.

There is general agreement that the polypteriform fishes, like Polypterus senegalus, constitute a unique lineage in the evolution of the vertebrates. However, the precise position of these fishes had been a point of controversy since the time of Darwin and Huxley. There is now consensus that the polypteriform fishes are members of superorder Actinopterygii. However, within the Actinopterygii, it is still debatable as to whether the polypteriform fishes are an early offshoot of the Actinopterygii or a more recent sister group to the sturgeon and other extant chondrostean fishes. In this study the sequence of proopiomelanocortin (POMC), the common precursor for the melanocortins and beta-endorphin, was used to evaluate the phylogenetic position of the polypteriform fishes relative to other bony fishes. 3(')RACE and 5(')RACE protocols were used to amplify overlapping regions of a POMC cDNA from the brain of P. senegalus. The full-length POMC cDNA had an open reading frame that encoded 259 amino acids. As seen in most gnathostomes, P. senegalus POMC has three melanocortin sequences (ACTH/alpha-MSH, gamma-MSH, and beta-MSH), and a beta-endorphin region. For phylogenetic analysis, the following POMC sequences were aligned at the amino acid level and analyzed using a maximum parsimony algorithm: P. senegalus, dogfish, sturgeon A, paddlefish A, sockeye salmon A, tilapia, and gar. The dogfish POMC sequence was used as the out-group. In this analysis the P. senegalus POMC sequence formed a clade with the chondrostean POMC sequences (sturgeon A and paddlefish A), and not with the neopterygian sequences (sockeye salmon A, tilapia, and gar). P. senegalus POMC is remarkably similar to sturgeon POMC A. In particular, in both precursors there is evidence for degeneration at the proteolytic cleavage site that precedes the gamma-MSH sequence. Based on the analysis of this nuclear gene it would appear that P. senegalus belongs to a branch of the chrondrostean lineage rather than representing a lineage of ray-finned fish that is ancestral to the chondrostean and neoptyergian ray-finned fishes. Alternatively, if the polypteriform fishes are in fact an early offshoot of the Actinopterygii (the traditional view), then the observations made for P. senegalus POMC relative to the chondrostean POMC sequences is the result of convergence.

Presence of the delta-MSH sequence in a proopiomelanocortin cDNA cloned from the pituitary of the galeoid shark, Heterodontus portusjacksoni.

Since a fourth MSH sequence, delta-MSH, has been detected in the proopiomelanocortin (POMC) gene of a dogfish and a stingray, members of superorder Squalea (class Chondrichthyes), it is possible that this novel MSH sequence might be a feature common to the POMC genes of all modern sharks and rays. As an initial step towards addressing this question, a full-length POMC cDNA was cloned and sequenced from the pituitary of the Port Jackson shark, Heterodontus portusjacksoni. The Port Jackson shark represents one of the oldest lineages in superorder Galea, and this superorder together with superorder Squalea form infraclass Neoselachii (the extant sharks and rays). The Port Jackson shark POMC cDNA has an open reading frame that is 1032 nucleotides in length and encodes the deduced amino acids sequences for beta-endorphin, ACTH/alpha-MSH, beta-MSH, gamma-MSH, and delta-MSH. Port Jackson shark delta-MSH has 83% primary sequence identity with dogfish and stingray delta-MSH, and it appears that the delta-MSH sequence may have been the result of an internal domain duplication and reinsertion of the beta-MSH sequence. The presence of the delta-MSH sequence in the POMC genes of representatives of both superorders of infraclass Neoselachii would indicate that the delta-MSH sequence must have been present in the ancestral euselachian shark that gave rise to the neoselachian radiation.

Analyzing the evolution of the opioid/orphanin gene family.

Advances in molecular biology have made it possible to rapidly obtain the amino acid sequence of neuropeptide precursors-either by cloning and sequencing the cDNA that encodes the precursor, or by reconstructing the arrangement of exons and introns in a neuropeptide-coding gene through genomic approaches. The databases generated from these molecular approaches have been used to design probes to identify the cells that express the gene, or to ascertain the rate of expression of the gene, and even to predict the post-translational modifications that can generate functional neuropeptides from a biologically inert precursor. Although the power of these approaches is substantial, it is appreciated that a gene sequence or an mRNA sequence reflects the potential products that may be assembled in a secretory cell. To understand the functional capabilities of the secretory cell, the molecular genetics approaches must be combined with procedures that actually characterize the end-products generated by the secretory cell. Recent advances in two-dimensional gel electrophoresis and mass spectrometry now make it possible to analyze neuropeptides from a relatively small amount of tissue. These procedures can reveal novel end-products, tissue-specific endoproteolytic cleavage events, and developmental shifts in post-translational processing schemes. A gene family that illustrates all of these processes and the advantages of combining genomics with proteomics is the opioid/orphanin gene family.