Epithelial sheet movement: effects of tunicamycin on migration and glycoprotein synthesis

Corneas with central epithelial wounds, 3 mm in diameter, were organ cultured in the presence of tunicamycin (TM) (1 microgram/ml), an antibiotic that inhibits glycosylation of asparagine-linked glycoproteins. Compared with control corneas, which healed in 22 hr, corneas cultured in the presence of TM for the entire culture time or for only the first 6 hr displayed a progressively slower epithelial healing rate that essentially dropped to zero by 24 hr of culture time. At 24 hr, approximately 75% of the wound was covered. After repeated washings with TM-free culture media (6X, 10 min each), this effect could consistently be reversed in corneas exposed to TM for 6 hr. Incorporation of [3H]glucosamine into trichloroacetic acid-precipitable proteins of migrating epithelial sheets was reduced to 14% that of controls after 12 hr of culture with TM, whereas [14C]leucine incorporation was not significantly affected. The decreased glycosylation was reflected on the cell surface after 12 and 20 hr culture in the presence of TM: apical cell membranes of the first six cells of the leading edge of the migrating sheet bound significantly fewer ferritin-concanavalin A particles per micrometer of membrane than did controls. These results indicate that synthesis of asparagine-linked glycoproteins is required for continued migration of corneal epithelial sheets. The asparagine-linked glycoproteins that are required for migration probably include cell-surface glycoproteins.     

Measurements of electron spin resonance with the pyruvate dehydrogenase complex from Escherichia coli. Studies on the allosteric binding site of acetyl-coenzyme A

Binding of the feedback inhibitor acetyl-coenzyme A to the pyruvate dehydrogenase complex from Escherichia coli was studied by electron spin resonance spectroscopy with the spin-labelled acetyl-CoA analogue 3-carboxy-2,2,5,5-tetramethylpyrrolidine-1-oxyl-CoA-thioester. The spin-labelled compound binds to the pyruvate dehydrogenase component of the enzyme complex and this binding can be reversed by acetyl-CoA, while CoA has no effect. AMP and fructose 1,6-bisphosphate, which are both activators of the pyruvate dehydrogenase complex, exhibit a partial competition with the spin-labelled acetyl-CoA analogue and it could be shown that both activators act essentially by reversion of the feedback inhibition of acetyl-CoA. The binding site for these activators seems to overlap with the acetyl-CoA binding site, possibly by a common phosphate attachment point. No competition for binding to the feedback inhibition site exists with pyruvate, thiamine diphosphate, magnesium ions and with the fluorescent chromophore 8-anilino-1-naphthalene sulfonic acid. Thus, the feedback inhibition site proves to be a true allosteric regulatory site, which appears to be completely separate from the catalytic site on the pyruvate dehydrogenase component. The spin-labelled acetyl-CoA analogue binds also to the product binding site of acetyl-CoA on the dihydrolipoamide acetyltransferase component of the pyruvate dehydrogenase complex. Two binding sites per polypeptide chain with identical affinities on this enzyme component were found and the binding of the analogue can be inhibited by acetyl-CoA as well as by CoA.     

H-2-linked genetic control of the plaque-forming cell response to sheep red blood cells

The role of genes linked to the H-2 locus in effecting an immune response to SRBC was examined using strains of mice which differ in the classes of antibodies produced following multiple injections with SRBC. While H-2-linked gene action appeared to be at the level of regulating the number of plaque-forming cells (PFC) present in the spleens of different strains following two injections with SRBC, non-H-2-linked immune response genes seemed to determine whether an IgM-IgG switch occurred as well as how much of each antibody class was produced by the number of PFC available as a result of H-2-linked gene intervention. Mapping studies showed that the H-2-linked genetic effects were due to either the requirement for two genes or the presence of genes located between I-B and H-2D.     

Genetic analysis of hypertropic mutants of Phycomyces

A new class of Phycomyces behavioral mutants with enhanced tropic responses has been analyzed genetically to determine the number of genes involved and the nature of their expression. These hypertropic mutants carry pleiotropic nuclear mutations. Besides their effects on sensory behavior, they also affect morphology and meiotic processes. Behavioral analyses of heterokaryons containing hypertropic and wild-type nuclei in varying proportions show that the hypertropic mutations in strains L82, L84, L86, and L88 are strongly dominant. Conversely, the hypertropic mutations carried by the strains L83, L85, and L87 are strongly recessive. We performed recombination analyses between hypertropic mutants and mutants with diminished phototropism, affected in the seven genes madA to madG. We found no evidence of linkage between the hypertropic mutations and any of these mad mutations. From crosses, we isolated double mutants carrying hypertropic mutations together with madC (night blind) and madG (stiff) mutations. The behavioral phenotypes of the double mutants are intermediate between those of the parentals. Complementation analyses show that the three recessive hypertropic mutations affect the same gene, which we call madH. The expression of the recessive hypertropic allele becomes dominant in heterokaryons carrying madC and madH nuclei; the madC gene has been implicated separately with the photoreceptor at the input to the sensory pathway, while the madH gene is associated with the growth control output. This result suggests the physical interaction of both gene products, madH and madC, in a molecular complex for the photosensory transduction chain.     

Tissue repair in a poikilothermic vertebrate, Carassius auratus (L.): a preliminary study

The effects of mechanical injury on the skin, connective tissue, muscle and bone of the goldfish is described 28 days following the injury. There is evidence of a dynamic response but the apparent inability of muscle regeneration to any degree is noted.