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Lipids tightly bound to cell and tissue proteins (lipoproteins) were investigated with the acetone-Sudan procedure in cryostat-cut sections of normal and regenerating limb tissues of the adult newt, Diemictylus viridescens.Nuclear and cytolplasmic membranes of all tissues stained moderately black; the nucleo-and cytoplasm stained less intensely than their membranes. Connective tissue fibers of the dermis, mysial and neural sheaths, and tunics of blood vessels yielded intense responses, In striated muscle of the limb, myofibrils and associated striations responded strongly. Nerve myelin responded weakly.In the preblastemic regenerate, the fibrous adepidermal basement membrane terminated abruptly at the surface of amputation. A distal fibrocellular residue was evident in continuity with the retrograde degeneration of amputation-injured muscle: observations suggest a possible contribution to the fibrocellular reticulum from myofibrils. The regeneration blastema appears isolated from proximal limb tissues by the intervening fibrocellular reticulum. The response of the blastema cell is relatively weaker than that of other cells and tissues. Regenerating muscle is recognized by the appearance of prominently stained myofibrils in myoblastic extensions off the limb stump musculature. The matrix and chondrocytes of regenerating cartilage stain for lipoprotein, with the osteoid of osteogenic centers responding strongly.Polychrome responses were obtained from hyalin-bodies within interphase nuclei, and from chromosomes in mitosis, suggesting that chromosomal lipid has been stained.Supporting the lipidic character of the observed responses is the negative reaction following long term lipid extraction in warm chloroform: methanol.Histochemical studies are currently being conducted on the lipids of the normal and regenerating tissues of the adult newt forelimb, based on the suggestion by Schmidt and Weidman ('64) that lipids may play a significant role in the events of regeneration. Previous studies have revealed a prominent sudanophilic lipid response (Schmidt, '66a), acidic and nonacidic lipid, phosphatides, and stainable cholesterol (Schmidt, '66b) in these tissues. However, the characterization of the tissue lipids, especially that of the regeneration blastema, remains incomplete. Therefore, we undertook the study of socalled masked lipids, or lipoproteins, in the normal and regenerating tissues of the adult newt limb: preliminary studies have been reported (Schmidt, '64).Lipids that are tightly bound to tissue proteins have been demonstrated by various histochemical procedures, e.g., Ackerman, '52; Berenbaum, '58; Ciacco, '34; Serra, '58; and others. These methods are founded on the prior or simultaneous application of some unmasking agent. Of the several possible procedures we employed, the lipoprotein responses which are reported herein are based exclusively on the acetone-Sudan test of Berenbaum ('58). Warm acetone serves as the unmasking agent and sudan black B as the lipid indicator. Although objections have ...
Lipids tightly bound to cell and tissue proteins (lipoproteins) were investigated with the acetone-Sudan procedure in cryostat-cut sections of normal and regenerating limb tissues of the adult newt, Diemictylus viridescens.Nuclear and cytolplasmic membranes of all tissues stained moderately black; the nucleo-and cytoplasm stained less intensely than their membranes. Connective tissue fibers of the dermis, mysial and neural sheaths, and tunics of blood vessels yielded intense responses, In striated muscle of the limb, myofibrils and associated striations responded strongly. Nerve myelin responded weakly.In the preblastemic regenerate, the fibrous adepidermal basement membrane terminated abruptly at the surface of amputation. A distal fibrocellular residue was evident in continuity with the retrograde degeneration of amputation-injured muscle: observations suggest a possible contribution to the fibrocellular reticulum from myofibrils. The regeneration blastema appears isolated from proximal limb tissues by the intervening fibrocellular reticulum. The response of the blastema cell is relatively weaker than that of other cells and tissues. Regenerating muscle is recognized by the appearance of prominently stained myofibrils in myoblastic extensions off the limb stump musculature. The matrix and chondrocytes of regenerating cartilage stain for lipoprotein, with the osteoid of osteogenic centers responding strongly.Polychrome responses were obtained from hyalin-bodies within interphase nuclei, and from chromosomes in mitosis, suggesting that chromosomal lipid has been stained.Supporting the lipidic character of the observed responses is the negative reaction following long term lipid extraction in warm chloroform: methanol.Histochemical studies are currently being conducted on the lipids of the normal and regenerating tissues of the adult newt forelimb, based on the suggestion by Schmidt and Weidman ('64) that lipids may play a significant role in the events of regeneration. Previous studies have revealed a prominent sudanophilic lipid response (Schmidt, '66a), acidic and nonacidic lipid, phosphatides, and stainable cholesterol (Schmidt, '66b) in these tissues. However, the characterization of the tissue lipids, especially that of the regeneration blastema, remains incomplete. Therefore, we undertook the study of socalled masked lipids, or lipoproteins, in the normal and regenerating tissues of the adult newt limb: preliminary studies have been reported (Schmidt, '64).Lipids that are tightly bound to tissue proteins have been demonstrated by various histochemical procedures, e.g., Ackerman, '52; Berenbaum, '58; Ciacco, '34; Serra, '58; and others. These methods are founded on the prior or simultaneous application of some unmasking agent. Of the several possible procedures we employed, the lipoprotein responses which are reported herein are based exclusively on the acetone-Sudan test of Berenbaum ('58). Warm acetone serves as the unmasking agent and sudan black B as the lipid indicator. Although objections have ...
Regenerating and non-regenerating limb tissues from the adult newt, Diemictylus viridescens, were assayed for isocitrate lyase activity. The enzyme assays were performed by micromodifications of existing procedures. In general, the whole homogenate, or a soluble fraction of the homogenate, was incubated with the substrate isocitrate. Isocitrate is cleaved by isocitrate lyase to glyoxylate and succinate. At the termination of the reaction, the 2,4-dinitrophenylhydrazone derivative of glyoxylate was produced, extracted and quantitated spectrophotometrically. Isocitrate lyase activity was localized to bulb and two-digit regenerates. The reaction product, glyoxylate (a monocarboxylic keto acid), was of special interest due to its role as a potent in vitro metabolic inhibitor. Therefore, the endogenous level of monocarboxylic keto acids, and the ability of regenerating tissue to produce and accumulate monocarboxylic keto acids, were investigated. Whole homogenates of regenerating tissue always contained more monocarboxylic keto acids than intact forearm or stump tissue.
Cryostat-cut sections of unamputated and amputated-regenerating limbs of the adult newt were examined following the Nile blue test for acidic and non-acidic lipids, the acid hematein and plasmal tests for phospholipids, and a Schultz test for cholesterol.Triglycerides (Nile blue test) are prominent in dermis and macrophages : triglyceride droplets are scattered in epidermis, wound epithelium, and regeneration blastema. Fatty acids (Nile blue test) are present in all tissues of the normal and regenerating limb: nerve myelin contains relatively little free fatty acid, while macrophages apFear to contain the least amount of this lipidic substance.Plasmalogens (plasmal test) are prominent in the myelin of nerves, and macrophages: a weak cytoplasmic reaction obtains in the epidermis, subcutaneous glands, striated muscle, tunics of blood vessels, wound epithelium, blastema cells, chondrocytes, perichondrium and periosteum.Mitochondria responding for cephalin, lecithin, and sphingomyelin (acid hematein test) are ubiquitously distributed among the cells and tissues of the normal and regenerating limb. These phosphatides are prominent in nerve myelin, macrophages, and in dermal droplets: a variable response obtains from the myofibrils of striated mu:cle.Cholesterol (Schultz test) was demonstrated only in nerve myelin and in macrophages associated witn injured nerves.Particular attention was paid to the lipid responses of the regeneration blastema, and the conclusion was reached that not all of the lipid previously demonstrated with sudan dyes was characterized by the current series of lipid tests.A modified Nile blue sulfate test that promises greater specificity in distinguishing between acidic and non-acidic lipids is introduced.
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