Activation and partial characterization of prolyl hydroxylase from regenerating limbs of the adult newt Notophthalmus viridescens

Prolyl hydroxylase activity extracted from regenerating newt (Notophthalmus viridescens) limb tissues can be increased by brief preincubation with cofactors (ascorbate, alpha-ketoglutarate, Fe2+ and O2) prior to assay with [3H]proline-labeled collagen substrate. Newt prolyl hydroxylase is optimally active at 30 degrees C, but loses activity rapidly at 37 degrees C. The presence of cofactors or substrate decreases enzyme heat lability. Detergents (Triton X-100 and octyl glucoside) do not aid enzyme extraction and inhibit enzyme activity. Activity solubilized during homogenization without detergent remains soluble following high speed centrifugations. Comparative studies are reported for enzyme extracted from chick embryo tissues.     

Increased prolactin binding and morphological changes in the wound epithelium of regenerating limbs of Notophthalmus viridescens

Summary Distribution of prolactin has been examined in regenerating forelimbs from the newt Notophthalmus viridescens. Specific prolactin binding was demonstrated in homogenates of unamputated tissue, and of regenerating limbs at from 3 to 21 days postamputation. Labeled prolactin that was injected intraperitoneally into animals with one regenerating limb accumulated in the most distal portion of the regenerate at 7 and 14 days postamputation. Light microscopic autoradiography demonstrated that labeled prolactin was localized most heavily in the apical, outer layer of the wound epithelium. Scanning electron microscopy demonstrated that, in addition to changes in prolactin affinity following amputation, morphological changes occurred in the apical wound epithelium as well. Cell surfaces of the stump epidermis were characterized by periodic dispersion of papillae among a network of interconnecting structures 1–2 m across. By contrast, the surfaces of cells from the area in which labeled prolactin was found to localize most intensely were characterized by lack of papillae and, depending on the stage of regeneration, a pattern of microvilli and microplicae. These morphological alterations appear to reflect functional and biochemical differences between stump epidermis and wound epithelium.     

Autonomic control of heart rate in the adult, aquatic Notophthalmus viridescens viridescens

1. We investigated the role of the autonomic nervous system in the control of the heart rate using an isolated heart preparation. 2. Addition of the parasympathetic blocker, atropine, to the organ bath resulted in an increase in heart rate as expected. 3. Addition of the sympathetic blocker, ergotamine, to the organ bath showed no change in the heart rate. 4. Addition of the sympathetic blocker, propranolol, to the organ bath resulted in the expected decrease in heart rate. 5. Both the sympathetic and parasympathetic nervous systems appear to play a role in the control of the heart rate.     

DNA synthesis in the thymus of the adult newt Notophthalmus viridescens.

Lymphocytic activity was examined in thymuses of adult newts by studying the number, location, morphology and fate of cells within thymuses which had been processed for autoradiography 15 min, 2 and 4 h, and 2, 4, and 10 days subsequent to the injection of tritiated thymidine. Results of this study indicate (1) that the adult thymus is a highly proliferative organ, (2) that large and medium-sized lymphocytes present in the peripheral parenchyma give rise to smaller lymphocytes which move centrally and emigrate from the thymus, and (3) that many thymocytes leave the thymus within 2-4 days after they have been produced. The significance of these findings is discussed.     

Vasculature in pre-blastema and nerve-dependent blastema stages of regenerating forelimbs of the adult newt, Notophthalmus viridescens

Immunocytochemistry utilizing a monoclonal antibody (BV1; blood vessel 1) highly reactive to the vasculature of the adult newt showed that a developing vasculature was present during early, pre-blastema, and early-bud blastema stages of forelimb regeneration in this species. Infusion of Prussian Blue and DiI into the brachial artery further delineated the intactness of this early vasculature. Finally, macroscopic observations of vascular flow underneath the apical epithelial cap (AEC) and microsurgical removal of the AEC and observation of subsequent bleeding buttressed the conclusion that an intact vasculature exists during early nerve-dependent stages of newt forelimb regeneration. The results suggest that this process of neovascular formation is angiogenesis, i.e., the formation of new vessels from pre-existing vessels in the stump. Furthermore, angiogenesis is an ongoing process initiated early after amputation. Blastema cells and the AEC are likely sourcesof factors that stimulate neovascularization.     

Comparison of the effects of actinomycin D on RNA synthesis of young and adult regenerating planarians

The regeneration of adult planarians is completed inhibited by antinomycin D (AMD), although that of young animals is slightly affected. The proposed hypothesis such as the required mRNA were available prior to section must be revised. In fact, RNA synthesis does occur in spite of AMD in the young. The dose effect, degree of inhibition and rapidity of effect of the inhibitor on RNA synthesis are compared at both ages.     

The sudanophilic lipid of normal and regenerating limb tissues of the adult newt,Diemictylus viridescens

The most intense and widely distributed sudanophilic responses of cryostat-sectioned newt limb tissues were obtained with a simultaneous fix and stain procedure of 1:1 10% formal-calcium and sudan black B. Droplets and globules of lipid mixtures and rodlets (mitochondria) were typical responses distributed within the epidermis, subcutaneous glands, dermis and other connective tissues, striated muscle (also with positive fibrils), tunics of blood vessels, and blood cells. A prominent droplet response was located subjacent to the adepidermal basement membrane. The myelin of brachial nerve stained intensely. In regenerating limbs, the wound epithelium response was comparable to that of epidermis. Post-amputational lipophanerosis of injured muscle and brachial nerves was observed. The retrograde degeneration of nerve myelin was extensive, and continued into the early differentiative phase of regeneration. Lipid-engorged macrophages were prominent among the injured tissues, distal to these, and within the wound epithelium. The regeneration blastema revealed a large quantity of sudanophilic lipid. Prominent droplet and rodlet responses were typical of the myelinating regenerating nerves. The response of regenerating muscle equaled that of the mature stump fibers. The cells of the regenerating chondroskeleton contained sudanophilic lipid. Organic solvents such as acetone, ether, chloroform and chloroform:methanol reduced or prevented the sudanophilic responses. Sudan red 7B revealed less lipid than did sudan black B. A fixation effect was demonstrated with post-chromated formalcalcium, and chromic-formalin fixed sections. In the latter preparations, swollen-bodies, identified as mitochondria, stained intensely.     

The antigenicity of regenerating tail tissue in the newt Diemictylus viridescens

Young adult male rabbits were inoculated with antigens prepared from regenerating (blastema stage) and nonregenerating tail tissues of the newtDiemictylus viridescens. Blood was collected from these rabbits after six weeks of semiweekly injection, two weeks of respite, and two more weeks of injections. A Freund adjuvant was added to the antigen preparations at the time of injection in order to elicit the anamnestic effect.Ouchterlony agar diffusions of the newt antigen preparations vs. the rabbit antisera were carried out. The resulting patterns of precipitation bands were compared and photographed.The strongest precipitation reactions of a given series were those between the antigen preparations made from nonregenerating tissue and their homologous antisera. The weakest reactions occurred between regenerating tissue antigens and regenerating tissue antisera. The strength of the antigen-antibody reactions was judged by the number of bands appearing in the diffusion plate and by the distinctness of these bands. Reactions of intermediate strength occurred between regenerating antigens and nonregenerating antisera, between nonregenerating antigens and regenerating antisera, and between antigens and antisera of different series.The loss of antigenicity during the blastemal period was considered to be related to the destruction of tissue in the wound areas at this time, and to indicate a quantitative rather than a qualitative loss of protein in regenerating tissue.This work is taken from data submitted to the Department of Biology of Fordham University in partial fulfillment of the requirements for the degree of Doctor of Philosophy, and was supported in part by a grant from the New York City Cancer Committee of the American Cancer Society.The author acknowledges his indebtedness to Dr.Alexander Wolsky of Fordham University, under whose direction this investigation was carried out, and Dr.John J. Corbett of Manhattan College and Lederle Laboratories.     

Factors altering DNA synthesis in the cardiac myocyte of the adult newt,Notophthalmus viridescens

To better understand the mechanisms governing the proliferation of cardiac myocytes it is important to identify the factors controlling this phenomenon, and to characterize their actions. DNA synthesis was quantified in vitro in ventricular myocytes from the adult redspotted newt, Notophthalmus viridescens. Ventricles were enzymatically separated and plated onto laminin. Myocytes were fed modified L-15 medium with 10% fetal bovine serum, and were variously treated with transforming growth factor-beta, transforming growth factor-beta combined with platelet-derived growth factor, acidic fibroblast growth factor, basic fibroblast growth factor, 12-0-tetradecanoylphorbol-13-acetate, heparin, or conditioned medium from ventricular myocytes or non-myocytes (primarily endothelial cells). With their final feeding the cells were given 1 Ci/ml of tritiated thymidine, and 24 hours later were fixed and stained. Dishes were coated with photographic emulsion, exposed, and developed. The percent of cells with labeled nuclei was determined. Experimental media that significantly increased DNA synthesis included those containing acidic fibroblast growth factor (121% of control), basic fibroblast growth factor (119% of control), 12-0-tetradecanoylphorbol-13-acetate (233% of control) and conditioned medium from ventricular myocytes (230% of control) or non-myocytes (128% of control). Media significantly inhibiting DNA synthesis were those containing heparin (31% of control), transforming growth factor-beta (38% of control), non-myocyte conditioned medium and heparin (75% of control), or transforming growth factor-beta and platelet-derived growth factor (63% of control).     

Hemopoietic tissue in the adult newt,Notopthalmus viridescens

The presence of developmental stages of lymphocytes and their precurors, as revealed by serial and thin sections of hemopoietic organs of normal adult newts (Notopthalmus viridescens) suggests that lymphopoiesis is limited to the thymus, medulla of the spleen and, to a lesser degree, the intestine. Stromal cells, small lymphocytes, granulocytes, mature erythrocytes and melanocytes were observed either within or near the parenchyma of the thymus. The urodele thymus differs from the thymus of anurans and higher vertebrates in that it lacks a cortex and a medulla, myoid cells and Hassall's corpuscles.     

Neural and hormonal stimulation of DNA and protein synthesis in cultured regeneration blastemata in the newt Notophthalmus viridescens

Distal portions of cone-stage newt forelimb blastemata were cultured transfilter to spinal ganglia for 36 or 72 hr. Addition of insulin to the medium consistently resulted in a significant increase (250% in ganglionated and 238% in nonganglionated blastemata) in the incorporation of [³H]thymidine into DNA, as compared to nontreated controls. When blastemata were cultured without ganglia for 36 or 72 hr, DNA synthesis decreased to 73 and 71%, respectively, of that achieved by ganglionated explants. When insulin was excluded from the medium, DNA synthesis decreased to 40% of insulin-treated explants, and, in the absence of both nerves and insulin, it declined to 31% of insulin-treated, innervated explants. The presence of insulin in the medium also resulted in an augmentation of (¹⁴C)-labeled amino acid incorporation into proteins; the average increase was 168%, as compared to untreated controls. l-thyroxine, growth hormone and hydrocortisone in combination with insulin, did not enhance the effects on DNA or protein synthesis of insulin alone. Also, exogenous cyclic nucleotides (cAMP, cGMP) and alterations of their endogenous levels with acetylcholine, sodium azide, theophylline or prostaglandins failed to elicit significant changes in DNA or protein synthesis. The existence of a synergistic action on DNA synthesis between nerves and insulin is suggested.     

Retinoic acid-induced pattern duplication in regenerating urodele limbs

The effects of varying doses of retinoic acid on forelimb regeneration in larval Ambystoma mexicanum amputated through the wrist joint and in adult Notophthalmus viridescens amputated through the basal carpals were compared. In both species, the major effect of retinoic acid was to cause the proximodistal duplication, in the regenerate, of stump segments proximal to the amputation plane. Transverse axial duplications (anteroposterior and dorsoventral) occurred in a smaller percentage of cases; these consisted of cartilage spurs in axolotls, and extra digits in newts. The frequency and magnitude of the proximodistal and (in the newt) transverse duplications were dose dependent, and the regenerating limbs were maximally sensitive to the retinoid during the period of dedifferentiation and accumulation of blastema cells. The effect of retinoic acid is exerted on cells local to the amputation surface, as shown by the fact that retinoic acid caused the proximodistal duplication of stump segments in regenerates derived from amputated distal lower arm segments grafted to the eyesocket.