Transferrin and the trophic effect of neural tissue on amphibian limb regeneration blastemas

Nerves promote regeneration of amputated urodele limbs, but the chemical basis of the effect is not known. We have examined the possible involvement of the iron-transport factor transferrin, which is important for cell proliferation and is present in vertebrate nervous tissue. Newt brain extract stimulated incorporation of [3H]thymidine in cultured blastemas from regenerating newt forelimbs, showing a biphasic dose-response similar to that of heterologous transferrin. As shown previously for transferrin, the inhibitory effect of brain extract at high concentrations was relieved by the addition of iron. Activity of brain extract was reduced by treatment with an iron-chelating agent and fully restored by the readdition of iron. Double immunodiffusion of newt tissue extracts and antibodies against newt plasma transferrin demonstrated the presence of transferrin-like factors in brain, spinal cord, and peripheral nerve. These results indicate that activity of transferrin may be part of the trophic effect of brain extract on cultured blastemas.     

Nerve dependent macromolecular synthesis in the epidermis and blastema of the adult newt regenerate

The present study is an analysis of neurotrophic control of DNA and protein synthesis in the separated epidermis and blastema of the early newt limb regenerate. Previous macromolecular studies employed the entire regenerate without discrimination between its two components. The results demonstrate that the specific activities of newly synthesized protein and DNA show a nerve dependence in both epidermis and blastema. There appears to be a postdenervation rise in synthesis followed by a decline to a plateau level in both components, similar to what is reported for the whole regenerate. After two days of denervation DNA synthesis in the mesenchymatous cells is more profoundly depressed, suggesting that the epidermis is less nerve dependent.     

P-epitope is characteristic for the neural tissue of vertebrates

In a search for antigens immunologically related to chordin, a notochord-specific glycoprotein of sturgeneous fishes, extracts from 55 samples of human and rabbit tissues were tested for inhibition of [125I]chordin binding to rabbit polyclonal antibodies. The strongest inhibition was observed with brain extracts of both species. Human, chicken, rabbit, and newt brain extracts also inhibited chordin binding in liquid phase to monoclonal antibodies (MAbs) against the P-epitope, the most immunogenic epitope of this glycoprotein. Immunohistochemical studies done on human and chicken embryos, newt, sterlet, and sturgeon embryos, larvae, and juveniles revealed a strong immunoreactivity of the brain, spinal cord, and tissue of the peripheral nervous system with an anti-P MAb. Other tissues, with several exceptions, showed a negative reaction in immunohistochemical experiments. The authors found that the P-epitope is ontogenetically expressed in the neural tissue of chicken, newt, and sterlet at the period of cytodifferentiation. Gel chromatography of human, chicken, and newt brain extracts showed that in each case the P-epitope was associated with a polydisperse macromolecular material of similar size. These antigens were designated as neurochordins. Prolonged pronase digestion of human and chicken brain extracts resulted in fragments with M about 3 kDa (presumably glycopeptides), which reacted with anti-P MAbs. These fragments were of the same size as corresponding glycopeptides of the pronase digest of chordin. Thus, in the present study, the P-epitope has been shown to be characteristic for the neural tissue of several vertebrate species; in the brain, it has been found in association with neurochordins, macromolecular antigens that are presumably protein conjugates with carbohydrates.     

Glial growth factor and nerve-dependent proliferation in the regeneration blastema of Urodele amphibians

After amputation of a limb from Urodele amphibians, division of the blastemal cells (the progenitor cells of the regenerate) depends on one or more unidentified growth factors provided by the nerve supply. Here we show that glial growth factor (GGF), a mitogenic protein previously purified from the bovine pituitary, is present in newt nervous system extracts. It is also detectable in extracts of the forelimb regeneration blastema, and its level there decreases after denervation. We have previously shown that blastemal cells dependent on the nerve for division are marked by a monoclonal antibody called 22/18. When denervated blastemas are cultured in the presence of partially purified GGF from newt brain, or pure GGF from the bovine pituitary, the thymidine labeling index of blastemal cells that are 22/18-positive is increased as much as sevenfold. These data indicate that GGF plays a role in nerve-dependent proliferation in the blastema.     

Newly-formed neurons in the regenerating optic tectum of Triturus cristatus carnifex

A histochemical light and electron microscopy study of acetylcholinesterase (AChE) was carried out on the regenerating opic tectum of adult newt. A plug of optic tectum was removed and 15 days later [6-H3] thymidine was injected. Ninety days after the lesion the brain was removed, treated for histochemical AChE-detection and autoradiographic analysis. This double treatment showed the capacity of these adult amphibians to regenerate the nervous tissue through the proliferation of undifferentiated elements and their subsequent differentiation into neurons as is shown by the presence of cells both labelled by [6-H3] thymidine and by the AChE-reaction product.     

Necessity of an adequate nerve supply for regeneration of the amputated pectoral fin in the teleost Fundulus

The present work deals with determination of the threshold of nerve fibers per unit of amputation surface necessary for regeneration of the pectoral fins of a teleost, Fundulus. Partial denervation of the amputated pectoral fins, i.e., resection of one or two of the three nerves of the brachial (=pectoral) plexus revealed that the presence of a single one allows the amputated fin to regenerate. From these data and others obtained previously, it is concluded that the nervous requirements for a teleost fin to regenerate are similar or slightly lower than those for tetrapods, for example in the newt, which are capable of appendage regeneration.     

Regeneration of the amphibian retina: role of tissue interaction and related signaling molecules on RPE transdifferentiation

Regeneration of eye tissue is one of the classic subjects in developmental biology and it is now being vigorously studied to reveal the cellular and molecular mechanisms involved. Although many experimental animal models have been studied, there may be a common basic mechanism that governs retinal regeneration. This can also control ocular development, suggesting the existence of a common principle between the development and regeneration of eye tissues. This notion is now becoming more widely accepted by recent studies on the genetic regulation of ocular development. Retinal regeneration can take place in a variety of vertebrates including fish, amphibians and birds. The newt, however, has been considered to be the sole animal that can regenerate the whole retina after the complete removal of the retina. We recently discovered that the anuran amphibian also retains a similar ability in the mature stage, suggesting the possibility that such a potential could be found in other animal species. In the present review article, retinal regeneration of amphibians (the newt and Xenopus laevis) and avian embryos are described, with a particular focus on transdifferentiation of retinal pigmented epithelium. One of the recent progresses in this field is the availability of tissue culture methods to analyze the initial process of transdifferentiation, and this enables us to compare the proliferation and neural differentiation of retinal pigmented epithelial cells from various animal species under the same conditions. It was revealed that tissue interactions between the retinal pigmented epithelium and underlying connective tissues (the choroid) play a substantial role in transdifferentiation and that this is mediated by a diffusible signal such as fibroblast growth factor 2. We propose that tissue interaction, particularly mesenchyme-neuroepithelial interaction, is considered to play a fundamental role both in retinal development and regeneration.     

Hormonal stimulation of cAMP-dependent protein kinase in rat pancreas

The phosphorylation of myelin (basic protein) purified from rabbit brain was markedly stimulated by exogenously added calmodulin in the presence of calcium and inhibited by W-7(N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide), a calmodulin interacting agent, in a dose-dependent fashion. However, exogenously added myelin basic protein free from protein kinase activity could not serve as a substrate of this calmodulin dependent protein kinase, suggesting that this kinase catalyzes the phosphorylation of the enzyme-substrate complex. These results suggest that a calmodulin-dependent protein kinase complex with the substrate (basic protein) is located in the myelin membrane of the central nervous system.     

Restricted inflammatory reaction in the CNS: a key impediment to axonal regeneration?

Axons in the central nervous system (CNS) of adult mammals do not regenerate after injury. Mammalian CNS differs in this respect from other mammalian tissues, including the peripheral nervous system (PNS), and from the CNS of lower vertebrates. In most parts of the body, including the nervous system, injury triggers an inflammatory reaction involving macrophages. This reaction is needed for tissue healing; when it is delayed or insufficient, healing is incomplete. The CNS, although needing an efficient inflammatory reaction resembling that in the periphery for tissue healing, appears to have lost the ability to supply it. We suggest that restricted CNS recruitment and activation of macrophages are linked to regeneration failure and might reflect the immune privilege that characterizes the mammalian CNS. As macrophages play a critical role in tissue restoration, and because their recruitment and activation are among the most upstream of the events leading to tissue healing, overcoming the deficiencies in these steps might trigger a self-repair process leading to recovery after CNS injury.     

CELL-FREE PROTEIN SYNTHESIS BY MOUSE BRAIN DURING EARLY DEVELOPMENT

—Cell-free homogenates were employed to study the nature of the mechanism that is responsible for the rapid decrement in protein synthesis during early neural development. There was a progressive loss of polypeptide synthesis in post-mitochondrial fractions that were isolated from increasingly older tissue. By the time the animals were approximately 17 days old, the rate of amino acid incorporation had decreased to the rate that was measured in adult brain preparations. This decrement in synthetic activity was similar to that previously measured in developing intact brain cells. The loss in protein synthesis was demonstrated to be independent of cellular membrane permeability and under the influence of intracellular control mechanisms. Although the nature of the control mechanism is still not clear, a lack of template RNA to direct protein synthesis was not the limiting factor in the decreased synthesis of the older brain preparations.     

Evidence for dedifferentiation and metaplasia in amphibian limb regeneration from inheritance of DNA methylation

Amphibian limb regeneration is a process in which it has been suggested that cells of one differentiated type may dedifferentiate and give rise to cells of another type in the regenerate. We have used two tissue-specific hypomethylations in the newt cardioskeletal myosin heavy chain gene as lineage markers to follow the fate of cells during limb regeneration. Analysis of genomic DNA from different muscle cell populations allowed the assignment of one marker to the muscle (Hypo A) lineage and the other, more tentatively, to the 'connective tissue' (Hypo B) component of muscle. The contribution to regenerated limb cartilage and limb blastemal tissue by cells carrying these markers was estimated by quantitative analysis of Southern blot hybridizations using DNA from regenerate tissues. The results are consistent with a contribution of cells from both muscle and connective tissue lineages to cartilage in regenerated limbs. In addition, removal of the humerus at the time of amputation (eliminating any contribution from pre-existing cartilage), has provided evidence for an increased representation of cells carrying the connective tissue marker in regenerate cartilage but did not affect the representation of cells carrying the muscle cell marker.     

An inhibitor of protein synthesis prepared by protease treatment of mouse cerebral cortex cells

A substance was isolated from mouse brain cortical tissue that inhibits both cell division and protein synthesis by cells in culture. The inhibitor was released from cerebral cortex tissue by mild protease treatment. A single exposure of cells to as little as 1.25 μg of the isolated material was sufficient to inhibit BHK-21 cell protein synthesis by 20%. Higher concentrations and continual exposure resulted in 87% reduction in protein synthesis. The inhibition was shown to be independent of amino acid uptake and most effective against primary mouse embryo fibroblasts and neonatal mouse brain cell suspensions. Cells previously adapted to culture or transformed cells derived from the nervous system were less affected by, or refractory to, the inhibitor. The substance was shown to be nondialyzable, relatively resistant to thermal inactivation and the inhibitor activity was not removed by chloroform extraction. Two active fractions were identified by Bio-Gel P-100 chromatography and the protein synthetic inhibitor was removed by affinity chromatography with $\text{Ulex}$$\text{europus}$ agglutinin.     

Nvbeta-actin and NvGAPDH as normalization factors for gene expression analysis in limb regenerates and cultured blastema cells of the adult newt, Notophthalmus viridescens

The red-spotted newt has the ability to fully regenerate complex structures by creating a pool of dedifferentiated cells that arise in response to tissue injury. An understanding of the mechanisms involved in the regenerative ability of the newt is limited by a lack of characterized assays. This deficiency includes the cloning and validation of housekeeping genes for normalizing gene expression data. We describe the cloning, characterization and real-time quantitative PCR evaluation of the normalization potential of the newt homologues of cytoplasmic beta-actin and GAPDH during newt limb regeneration and within the blastemal B1H1 cell line. Nvbeta-actin demonstrates a heterogeneous expression during limb regeneration and may be associated with differentiation state. The level of Nvbeta-actin expression in B1H1 cultures under conditions of myogenesis and serum resupplementation varies with the treatment. NvGAPDH is ubiquitously expressed during limb regeneration and within B1H1 cultures and does not demonstrate overall variations in expression levels. Thus, NvGAPDH is a more appropriate normalization factor in gene expression analyses during limb regeneration and treatments of B1H1 cultures.     

EFFECTS OF ACUTE HYPERTHERMIA ON POLYRIBOSOMES, IN VIVO PROTEIN SYNTHESIS AND ORNITHINE DECARBOXYLASE ACTIVITY IN THE NEONATAL RAT BRAIN

—Acute hyperthermia produces in situ disaggregation of brain polyribosomes in infant rats, as determined by electron microscopy. Protein synthesis is inhibited in infant, but not weanling, rat brain by 45 min of hyperthermia; this inhibition is reversed during a 2 h recovery period at normothermic conditions. Hepatic protein synthesis was inhibited less than that of brain. Acute hyperthermia also leads to a profound loss of ornithine decarboxylase activity in brain; during recovery the activity of this enzyme overshoots to values greater than those of normothermic control rats. This increase is blocked by cycloheximide administration. In testis, a tissue with high ornithine decarboxylase activity, enzyme activity was not affected by hyperthermia and recovery, indicating tissue specificity for these effects.     

The short-term and long-lasting changes in DNA and RNA synthesis in the cerebral cortex cells of animals subjected to hypoxia and nerve tissue transplantation]

The DNA and RNA synthesis in the cells of the brain cortex of intact rats and animals subjected to hypoxia, hypoxia with subsequent transplantation or by the local brain injury has been investigated. The DNA synthesis changes insignificantly in the case of hypoxia, it enhances slightly in the area of the injury and increases much more after transplantation. The RNA synthesis decreases considerably immediately after hypoxia and decreases much more 120 days later. Using the ultracentrifuge method it has been found that under the effect of hypoxia the number of nervous cells decreases, the number of glial cells does not change. The local injury in the nervous tissue enhances abruptly the synthesis in neurons and glial cells in the hypoxia-exposed animals, the embryonic nervous tissue transplantation normalizes the number of neurons in the specimens under study and the RNA synthesis in the neurons and glial cells.     

On the Nature of the Neurotrophic Phenomenon in Urodele Limb Regeneration

The nervous control of regeneration of body parts in the urodeleamphibian and other animals has been one of the best model systemsfor the study of the neurotrophic phenomenon. In the past mostof these studies were experimental morphological, but recentlythe salient problems on the nature of the cellular responseto the neurotrophic agent and the nature of the nervous agentitself are also analyzed molecularly. The ensemble of studiesreviewed in the present work, which also show that the agentof the nerve is a peptide and defines aspects of its effecton molecular synthesis in regenerate cells, leads me to advancethe following theories. I propose that the neurotrophic agentaffects only the rate of ongoing events in the cell and notthe quality and kind of the events; that the events are alreadyindigenous to the responding cells; that alteration in therateof events, for example increasing the rate of molecular syntheses,yields an increased cell population which by its size and increasedcellular interactions has formative and differentiated capabilitieswhich do not exist in a smaller cell population; and finallythat the neurotrophic factor (NTF) is one of many "conversational"peptides including nervegrowth factor (NGF) and epidermal growthfactor (EGF) which function to alter the absolute rate of ongoingcellular events.     

Assay of cyclic 3'-5'-monophosphate in the regenerating forelimb of the newt, Triturus.

The cyclic adenosine 3′-5′-monophosphate content of four regenerate stages in the forelimb of the newt, Triturus viridescens, was assayed using the Gilman method and compared to the content in the normal, unamputated, forelimb. The concentration was found to be highest in the earliest stages of regeneration, followed by a sharp drop and then a rise to a plateau approximately that of the unamputated limb. The possibility that cyclic AMP acts as a second messenger for nervous and hormonal influences on regeneration is discussed.     

Specific Lymphocyte Sensitization in Cancer: Is There a Common Antigen in Human Malignant Neoplasia?

From human cancer tissue a basic protein can be extracted by the method which yields encephalitogenic factor when applied to human brain. This tumour basic protein (obtained from several different neoplasms) acts as an antigen in the cytopherometric test for malignant neoplasia and in general gives higher results than does brain basic protein. The reverse is true when degenerative disease of the nervous system is studied. The basic protein extractable from brain and from tumours thus has some degree of specificity probably referable to its amino-acid sequence.