Carbamylcholine, TRH, PGF2 alpha and fluoride enhance free intracellular Ca++ and Ca++ translocation in dog thyroid cells

Effects on Ca++ translocation and [Ca++]i were studied in dog thyro?d cell monolayers using both 45Ca++ efflux and the indicator quin-2. Carbamylcholine, a non hydrolysable analog of acetylcholine, through muscarinic receptors, and to a lesser extent TRH and PGF2 alpha increased both these parameters. [Ca++]i increased by 171, 100 and 75% respectively over a basal level of 66 +/- 17 nM (mean +/- SD). The response to carbamylcholine was biphasic. A transient increase in [Ca++]i was followed by a more sustained phase where the [Ca++]i was slightly higher than the basal level. Only the first phase was insensitive to extracellular Ca++ depletion. This phase is probably due to a release of Ca++ from an intracellular store. NaF also induced a sustained rise in [Ca++]i dependent on extracellular Ca++ and affected 45Ca++ efflux. Our data provide direct evidence of an implication of intracellular Ca++ in the response of dog thyro?d cells to all these agents.     

Constructional morphology: The analysis of constraints in evolution dedicated to A. Seilacher in honour of his 60. birthday

Evolutionary change is opportunistic, but its course is strongly constrained in several fundamental ways. These constraints (historical/phylogenetic, functional/adaptive, constructional/morphogenetic) and their dynamic relationships are discussed here and shown to constitute the conceptual framework of Constructional Morphology. Notwithstanding recent published opinions which claim that the discovery of constraints renders Neodarwinian selection theory obsolete, we regard the insights of Constructional Morphology as being entirely consistent with this theory. As is shown here in the case of the Hyracoidea, formal analysis of the constraints which have framed the evolution of various characters extends our understanding of the evolution of a taxon.     

Antagonistic effects of thyrotropin and epidermal growth factor on thyroglobulin mRNA level in cultured thyroid cells

Both thyrotropin (TSH) and epidermal growth factor (EGF) are potent mitogenic agents when added to dog thyroid cells in primary culture [Roger, P. P. and Dumont, J. E. (1984) Mol. Cell. Endocrinol. 36, 79-93]. The concomitant effect of these agents on the differentiation state of the cells was appreciated using cell morphology, iodide trapping, thyroglobulin synthesis and cytoplasmic thyroglobulin mRNA content as markers. Together with previous results [Mol. Cell. Endocrinol. 36, 79-93 (1984)] it is shown that cells cultured in the continuous presence of TSH maintain all the parameters at a near normal level. In the absence of TSH, thyroglobulin mRNA decreased to very low, though still detectable levels. Addition of TSH restored subnormal mRNA levels. Culture of cells in the presence of EGF for 4-6 days affected profoundly their morphology, abolished iodide trapping and decreased thyroglobulin synthesis and cytoplasmic mRNA content to undetectable levels. Addition of TSH to cells previously exposed to EGF reversed the growth factor effect on all four indexes. The redifferentiating effect of TSH was well observed within 3-4 days and was mimicked by the adenylate cyclase activators, forskolin and cholera toxin. When administered simultaneously, TSH and EGF achieved an intermediate situation, EGF antagonizing partially the effect of TSH on the expression of thyroglobulin gene. Another growth factor, fibroblast growth factor, while promoting thyroid cell proliferation also, did not interfere at all with TSH effects on cytoplasmic thyroglobulin mRNA content. Our results make the dog thyroid cell in primary culture an appropriate model to study the mechanisms involved in gene regulation by cyclic AMP and growth factors.     

(AT)n is an interspersed repeat in the Xenopus genome.

We have observed (AT)34 and (AT)23 tracts close to the coding sequences of the Xenopus laevis tadpole alpha T1 and adult beta 1 globin genes, respectively. We show that (AT)n sequences are found as interspersed repeats within the Xenopus globin and histone gene loci. Using (AT)n co-polymer in filter hybridisation experiments we estimate that there are 10(4) (AT)n tracts per haploid Xenopus genome. Hybridisation to genomic blots of DNA from yeast, slime mold, trypanosome, fruit fly, salmon, chicken, rat, human, crab and Xenopus species shows that strictly alternating AT of sufficient length to hybridise appears to be most abundant in Xenopus and crab genomes. We show that the specificity of the co-polymer probe for strictly alternating AT is, however, dependent on the length of the probe. Hybridisation experiments using (TG)n copolymer suggest that this highly conserved repeat is found as clustered repeats in the Xenopus genome in contrast to other eukaryotic genomes so far studied.     

Migration of peripheral T and B cells into the thymus of aging (NZB × SJL)F1 female mice

Aging NZB × SJL (NS) female mice provide a unique model of thymopathology characterized by the intrathymic accumulation of large numbers of mature T and B cells. The purpose of the present work was to examine the possibility that this phenomenon results from the invasion of the thymus by cells from the periphery. Lymphoid cells labeled with chromium-51 or indium-111 were injected into syngeneic recipients to study their patterns of in vivo migration. Lymph node (LN) or spleen cells were found to localize significantly (1–2% of injected radioactivity) into the thymus of 12-month-old NS females but not into that of young recipients or of old NS males. However, intrathymic localization of injected LN cells was observed in castrated NS males which exhibit the same thymopathology as NS females. Both radiolabeled T and B cells were found to enter the thymus of aged NS females but the latter cells about three times less efficiently than the former. Moreover, while thymocytes from young NS females were unable to recirculate to LN, those of old NS females showed increased LN-seeking capacity and part (1%) of them did migrate back into the thymus of old but not young NS females. In additional cell transfer experiments, the intrathymic migration of B cells into old NS females was further documented by using the antibody response to sheep erythrocytes as a tracer. Taken together, these observations indicate that the thymus of aging NS female mice is permeable to recirculating lymphocytes, suggesting that at least part of the mature T and B cells detected in this thymus, are migrants from the periphery.     

Identification of the dye gene product, mutational loss of which alters envelope protein composition and also affects sex factor F expression in Escherichia coli K-12

Summary The product of the dye gene of Escherichia coli, mapping at 99–100 min, is required for expression of the sex factor F, and also appears to be involved in the regulation of envelope proteins. Mutation of dye thus results in loss of expression of the F-factor (Fex^–, i.e. male sterility, and dye sensitivity (Dye^s). We have isolated a plasmid, pRB38, in which a 6 kb SalI fragment carrying the dye ⁺ gene was cloned into the plasmid pACYC184. This 6 kb SalI fragment also carries two nearby markers, chlG, involved in the synthesis of the molybdenum cofactor, and phoM, required for constitutive expression of alkaline phosphatase.Some of the polypeptides synthesised by pRB38 were identified using the maxi-cell procedure. The product of the dye gene was found to be a polypeptide of M_r=29,000. Thus derivatives of pRB38 in which the transposon was inserted into dye, resulting in a Dye^S Fex^– phenotype when these plasmids were in a dye strain, failed to produce this polypeptide and in some cases produced a truncated product. Such insertions also resulted in a Chl^r and Pho^– phenotype when the plasmid was in a (dye-chlG-phoM) phoR strain, although complementation tests suggested that the phoM ⁺ and chlG ⁺ genes were still intact. Insertions of into the promoter distal end of dye did not result in a Dye^S Fex^– phenotype, although a truncated Dye protein was synthesised, and a Chl^r Pho^– phenotype was produced.It has been suggested (Gaffney et al. 1983) that the dye (=sfrA) gene product is necessary for F-factor expression because it is required for translocation of the F-factor TraJ protein to the outer membrane. Our results suggest that the Dye protein is also required for expression of the molybdenum cofactor and of alkaline phosphatase, and could perhaps be involved in the translocation of these proteins to the membrane.     

Structural analysis of a maize gene coding for glutathione-S-transferase involved in herbicide detoxification

Summary We have used the cDNA clone encoding maize glutathione-S-transferase (GST I) to isolate a genomic DNA clone containing the complete GST I gene. Nucleotide sequence analysis of the cDNA and genomic clones has yielded a complete amino acid sequence for maize GST I and provided the exon-intron map of its gene. The mRNA homologous sequences in the maize GST I gene consist of a 107 bp 5 untranslated region, a 642 bp coding region and 340 bp of the 3 untranslated region. They are divided into three exons by two introns which interrupt the coding region. The 5 untranslated spacer contains an unusual sequence of pentamer AGAGG repeated seven times. The inbred maize line (Missouri 17) contains a single gene for GST I, whereas the hybrid line (3780A) contains two genes. Nucleotide sequence analysis of the primer extended cDNA products reveals that the 5 untranslated regions of the two genes in the hybrid 3780A are identical except for a 6 bp internal deletion (or insertion). The amino acid sequence of maize GST I shares no apparent sequence homology with the published sequences of animal GST's and represents the first published sequence of a plant GST. re]19850813 ac]19851126     

Chronic toxicity of three insecticides (carbaryl,fenthion and lindane) in the freshwater snail Lymnaea stagnalis

A chronic intoxication with carbaryl, fenthion and lindane was induced in young snails. The parameter k of the von Bertalanffy's equation showed clearly the growth changes induced by these insecticides. In all cases the fecundity of intoxicated snails was reduced. Among these three insecticides, lindane was the most toxic, carbaryl the least.     

Identification of specific proteins and glycoproteins associated with membrane fractions isolated from Zea mays L. coleoptiles

The aim of this work was to identify proteins specific for plant cell membranes which could then be used as unique markers. A crude membrane fraction was isolated from corn coleoptiles and separated on non-linear sucrose density gradients. Separation of endoplasmic reticulum (NADH-cytochrome c reductase), mitochondria (cytochrome c oxidase), golgi (inosine diphosphatase), and plasma membranes (N-1-naphthylphthalamic acid-binding) was achieved. The membrane proteins from the gradient fractions were separated using sodium dodecyl sulphate-poly-acrylamide gel electrophoresis and the gels stained with coomassie blue or with concanavalin A/peroxidase to detect glycoproteins. Proteins specific for the various membranes were identified. Five proteins including two glycoproteins were plasma membrane markers. Protoplasts were isolated and iodinated using lactoperoxidase/glucose oxidase covalently attached to beads. Eleven iodinated proteins were found and three of these corresponded to proteins specifically associated with plasma membranes in the density gradients. Two methods for detecting Ca²⁺-binding proteins following sodium dodecylsulphate polyacrylamide gel electrophoresis were employed. The majority of such proteins were found in the endoplasmatic reticulum and one was specific for plasma membranes. In vitro and in vivo phosphorylation of membrane proteins was examined and the majority of proteins phosphorylated were glycoproteins. Two of the phosphorylated proteins (M_r=110,000 and 20,000) were also iodinated on protoplasts and may be part of the plasma membrane ATPases.Abbreviations ER endoplasmic reticulum - IDP inosine diphosphate - NPA N-1-naphthylphthalamic acid     

Use of 6-diazo-5-oxo-l-norleucine to study interaction between myocardial glycoconjugate secretion and endothelial activation in the early embryonic chick heart

The glutamine analog, 6-diazo-5-oxo-l-norleucine (DON), a glycoconjugate inhibitor, was used to probe the relationships between myocardial secretion of extracellular matrix and endothelial differentiation and formation of cushion mesenchyme (primordia of AV values). When DON was given to stage 12 chick embryos maintained in shell-less culture, the myocardial secretion gradient of glucose- and sulfate-labeled matrix was blocked. Concomitantly, the endothelium failed to complete activation but continued to divide and incorporate thymidine. By varying DON concentration, two distinct phases of endothelial differentiation were identified: the first (labile to 0.5 μg) involved hypertrophy, the second (labile to 0.25 μg) acquisition of migratory appendages with resultant mesenchyme formation. Glucosamine + DON (but not inosine, glucose, or glutamine) restored the matrical secretion gradient and to varying degrees both phases of endothelial activation. Endothelia totally suppressed from forming mesenchyme in situ acquired this capacity when explanted into three-dimensional collagen gel culture. The capacity was enhanced by glucosamine given in situ as an inhibitory override, dependent upon serum concentration, inhibited by heat-inactivated serum or by adding DON to the medium, but unaffected by hyaluronate. These results were compared to those obtained by co-culturing endothelium and myocardium and discussed in terms of the hypothesis that cushion mesenchyme formation results from an epithelial interaction mediated by glycoconjugates.