An investigation of the prolactin-thyroxine synergism in newt limb regeneration

The use of hormone replacement to support limb regeneration in hypophysectomized newts has been the subject of many investigations. Growth hormone, as well as prolactin (PL) in combination with exogenously supplied thyroxine, have all been shown to he effective. However, the bovine growth hormone used to support limb regeneration was contaminated by prolactin and thyroidstimulating hormone (TSH). The present investigation evaluates the significance of (1) prolactin contamination and (2) endogenous thyroxine synthesis resulting from TSH contamination on limb regeneration in hypophysectomized newts. The effect of supplying exogenous thyroxine was also evaluated. Our studies showed that when hypophysectomized newts were injected with contamination levels of PL and TSH, regeneration occurred, suggesting that the newt's thyroid synthesized sufficient thyroxine to support a prolactin-thyroxine synergism. The endogenous thyroxine was synthesized by thyroid glands that were indistinguishable from those of saline-injected, hypophysectomized controls.     

Isolation of isoflavones from soy-based fermentations of the erythromycin-producing bacterium Saccharopolyspora erythraea

A search for an abundant and economical source of isoflavones, particularly genistein, led to the discovery that the erythromycin-producing organism Saccharopolyspora erythraea also produces this promising new cancer-prevention agent. Erythromycin fermentation is a large-scale, soybean-based process used world-wide for the commercial production of this medically important antibiotic. Results from this study indicate that genistin (the glucoside form of genistein), which is added to the fermentation in the soybean media, was converted to genistein through the action of a β-glucosidase produced by the organism. Genistein was co-extracted with erythromycin from the fermentation broth, then separated from erythromycin during the second step of the purification process for the production of erythromycin. Received 10 September 1996 / Received revision: 22 November 1996 / Accepted: 7 December 1996     

Low density lipoprotein cytotoxicity induced by free radical peroxidation of lipid

Low density lipoprotein (LDL) has been reported to be injurious or toxic to cells in vitro. This injurious effect is, in some instances, due to oxidation of the lipid moiety of the lipoprotein. The objectives of this study were to determine if the oxidation rendering the lipoprotein toxic to human skin fibroblasts occurred by free radical mechanisms, and if so, which of the common free radical oxygen species were involved. The selective free radical blockers or scavengers employed included superoxide dismutase for superoxide, catalase for hydrogen peroxide, dimethylfuran for singlet molecular oxygen, and mannitol for hydroxyl radical. The presence during lipoprotein preparation of general free radical scavengers (vitamin E, butylated hydroxytoluene) or the divalent cation chelator ethylenediamine tetraacetic acid prevented the formation of cytotoxic low density lipoprotein, while the simultaneous presence of superoxide dismutase and catalase partially inhibited its formation. The results indicate that superoxide and/or hydrogen peroxide are involved in the formation of the toxic LDL lipid. The toxic action of oxidized LDL could not be prevented by inclusion of antioxidants in the culture medium, indicating that an oxidized lipid was responsible for cell injury rather than free radicals generated in culture by the action of oxidized LDL. Three separate assays for cell injury (enumeration of attached cells, cell loss of lactate dehydrogenase into the culture medium, and trypan blue uptake) indicated a sequence of events in which the fibroblasts are injured, die, and then detach.     

Acridine Sensitivity of Bacteriophage T2H in Escherichia coli

Normally acridine-sensitive, Escherichia coli-T2H complexes are rendered acridine-resistant if the infecting bacteriophage mutant is either pr or q. If these pr or q mutants are treated to produce sensitive revertants, one obtains a mutation at any of several dye-sensitizing (ds) sites in the early enzyme region of the T2 map. The ds mutants are nonspecific suppressors because they reduce the resistance of complexes containing either pr or q to proflavine. The ds mutants are not identical in action, since some make pr or q sensitive to proflavine and quinacrine, and others, to proflavine alone. Two ds mutants have r to r(+) mutation patterns which differ, depending upon whether or not the ds is coupled with r7 (an rII mutant). The mutation patterns of r(+) to r are the same for both ds mutants and for wild type. We suggest that dye sensitization may consist of alterations of early enzymes so as to produce slightly different forms of deoxyribonucleic acid which are in turn dyesensitive.     

Goldfish cones secrete a two-repeat interphotoreceptor retinoid-binding protein

Vitamin A and fatty acids are critical to photoreceptor structure, function, and development. The transport of these nutrients between the pigment epithelium and neural retina is mediated by interphotoreceptor retinoid-binding protein (IRBP). IRBP, a 133-kDa (human) glycolipoprotein, is the major protein component of the extracellular matrix separating these two cell layers. In amphibians and mammals, IRBP consists of four homologous repeats of about 300 amino acids which form two retinol and four fatty acid-binding sites. Here we show that IRBP in teleosts is a simpler protein composed of only two repeats. Western blot analysis shows that goldfish IRBP is half the size (70 kDa) of IRBP in higher vertebrates. Metabolic labeling studies employing Brefeldin A taken together with in situ hybridization studies and the presence of a signal peptide show that goldfish IRBP is secreted by the cone photoreceptors. The translated amino acid sequence has a calculated molecular weight of 66.7 kDa. The primary structure consists of only two homologous repeats with a similarity score of 52.5%. The last repeats of human and goldfish IRBPs are 69.1% similar with hydrophobic regions being the most similar. These data suggest that two repeats were lost during the evolution of the ray-finned fish (Actinopterygii), or that the IRBP gene duplicated between the emergence of bony fish (Osteichthyes) and amphibians. Acquisition of a multirepeat structure may reflect evolutionary pressure to efficiently transport higher levels of hydrophobic molecules within a finite space. Quadruplication of an ancestral IRBP gene may have been an important event in the evolution of photoreceptors in higher vertebrates. Correspondence to: F. Gonzalez-Fernandez     

The pericentriolar material in Chinese hamster ovary cells nucleates microtubule formation

The structure and function of the centrosomes from Chinese hamster ovary (CHO) cells were investigated by electron microscopy of negatively stained wholemount preparations of cell lysates. Cells were trypsinized from culture dishes, lysed with Triton X-100, sedimented onto ionized, carbon-coated grids, and negatively stained with phosphotungstate. The centrosomes from both interphase and dividing cells consisted of pairs of centrioles, a fibrous pericentriolar material, and a group of virus-like particles which were characteristic of the CHO cells and which served as markers for the pericentriolar material. Interphase centrosomes anchored up to two dozen microtubules when cells were lysed under conditions which preserved native microtubules. When Colcemid-blocked mitotic cells, initially devoid of microtubules, were allowed to recover for 10 min, microtubules formed at the pericentriolar material, but not at the centrioles. When lysates of Colcemid-blocked cells were incubated in vitro with micotubule protein purified from porcine brain tissue, up to 250 microtubules assembled at the centrosomes, similar to the number of microtubules that would normally form at the centrosome during cell division. A few microtubules could also be assembled in vitro onto the ends of isolated centrioles from which the pericentriolar material had been removed, forming characteristic axoneme- like bundles. In addition, microtubules; were assembled onto fragments of densely staining, fibrous material which was tentatively identified as periocentriolar material by its association of CHO can initiate and anchor microtubules both in vivo and in vitro.     

Limb regeneration following the discontinuation of growth hormone therapy in the hypophysectomized newt,Notophthalmus viridescens

Untreated adult newts do not undergo normal limb regeneration following hypohysectomy. A fibrocellular dermal barrier (cicatrix) atypically forms between the apical epithelium and the underlying mesenchymal tissues. Historically, continuous administration of growth hormone or of prolactin in combination with thyroxine restored regenerative capacity to these newts. In a previous investigation, we demonstrated that the initial effect of these two hormone treatments, when administered on alternate days to hypophysectomized newts beginning eight days post-amputation, was to facilitate the erosion of the fibrocellular barrier and establish the epithelial mesenchymal interface that is observed in a regenerating limb. The present investigation was designed to evaluate the necessity of continuous hormone therapy to maintain limb regeneration in hypophysectomized newts. One, two, or three injections of growth hormone or of prolactin in combination with thyroxine was administered on successive alternate days to hypophysectomized newts either immediately following limb amputation (ID) or beginning eight days post-amputation (DD). The ID and DD newts receiving one, two, or three injections of growth hormone showed evidence of regeneration to the digitiform stage by day 30 post-amputation, while those receiving prolactin and thyroxine underwent wound healing. While both hormone treatments initially promoted a dermis-free apical epithelium, only hypophysectomized newts that had received growth hormone were able to continue regenerating. We have, therefore, concluded that discontinuous growth hormone therapy is sufficient to initiate and maintain the conducive environment for limb regeneration to advanced stages in the hypophysectomized newt. While initiating this process, prolactin and thyroxine therapy on a discontinuous regime does not maintain regeneration. The direct and indirect role of growth hormone in supporting limb regeneration in normal and hypophysectomized newts is discussed.     

The ultrastructure of the striated muscles of Derocheilocaris typica (Mystacocarida,Crustacea)

The striated muscles of Derocheilocaris typica consist of mononucleated cells, each containing one filament bundle. Large muscles consist of two or more cells adjacent to each other. The mitochondria line up along the filament bundle on one side. The nucleus is situated in the mitochondrial row and has a small cytoplasmic area around it filled with glycogen. The sarcomeres are between 3 and 6 μm long. The Z-line and H band are present. Six thin filaments surround one thick filament. All muscles belong to the phasic type. The tubular system emanates from the ends of the muscle cell and penetrates the whole cell. The tubules are formed as cisterns, which also open at the cell membrane at the level of the I bands. They have sarcoplasmic cisterns on both sides forming a continuous triad system. Partially transformed epidermal cells mediate muscle insertions on the cuticle. Tendons are formed with the transformed epidermal cells being supplemented by fibroblasts forming collagen fibers. Dorsal and ventral abdominal muscles are innervated from the dorso-lateral nerve arising from the nerve chain. Each muscle cell receives one axon, which forms one synapse on the mitochondrial-free side of the muscles. Axons form terminal spines, which make axo-axonal synapses.     

Lithium Fluoride Based Electron Contacts for High Efficiency n‐Type Crystalline Silicon Solar Cells

Low‐resistance contact to lightly doped n‐type crystalline silicon (c‐Si) has long been recognized as technologically challenging due to the pervasive Fermi‐level pinning effect. This has hindered the development of certain devices such as n‐type c‐Si solar cells made with partial rear contacts (PRC) directly to the lowly doped c‐Si wafer. Here, a simple and robust process is demonstrated for achieving mΩ cm² scale contact resistivities on lightly doped n‐type c‐Si via a lithium fluoride/aluminum contact. The realization of this low‐resistance contact enables the fabrication of a first‐of‐its‐kind high‐efficiency n‐type PRC solar cell. The electron contact of this cell is made to less than 1% of the rear surface area, reducing the impact of contact recombination and optical losses, permitting a power conversion efficiency of greater than 20% in the initial proof‐of‐concept stage. The implementation of the LiF_x/Al contact mitigates the need for the costly high‐temperature phosphorus diffusion, typically implemented in such a cell design to nullify the issue of Fermi level pinning at the electron contact. The timing of this demonstration is significant, given the ongoing transition from p‐type to n‐type c‐Si solar cell architectures, together with the increased adoption of advanced PRC device structures within the c‐Si photovoltaic industry.