Dapper Antagonist of Catenin-1 Cooperates with Dishevelled-1 during Postsynaptic Development in Mouse Forebrain GABAergic Interneurons

Synaptogenesis has been extensively studied along with dendritic spine development in glutamatergic pyramidal neurons, however synapse development in cortical interneurons, which are largely aspiny, is comparatively less well understood. Dact1, one of 3 paralogous Dact (Dapper/Frodo) family members in mammals, is a scaffold protein implicated in both the Wnt/β-catenin and the Wnt/Planar Cell Polarity pathways. We show here that Dact1 is expressed in immature cortical interneurons. Although Dact1 is first expressed in interneuron precursors during proliferative and migratory stages, constitutive Dact1 mutant mice have no major defects in numbers or migration of these neurons. However, cultured cortical interneurons derived from these mice have reduced numbers of excitatory synapses on their dendrites. We selectively eliminated Dact1 from mouse cortical interneurons using a conditional knock-out strategy with a Dlx-I12b enhancer-Cre allele, and thereby demonstrate a cell-autonomous role for Dact1 during postsynaptic development. Confirming this cell-autonomous role, we show that synapse numbers in Dact1 deficient cortical interneurons are rescued by virally-mediated re-expression of Dact1 specifically targeted to these cells. Synapse numbers in these neurons are also rescued by similarly targeted expression of the Dact1 binding partner Dishevelled-1, and partially rescued by expression of Disrupted in Schizophrenia-1, a synaptic protein genetically implicated in susceptibility to several major mental illnesses. In sum, our results support a novel cell-autonomous postsynaptic role for Dact1, in cooperation with Dishevelled-1 and possibly Disrupted in Schizophrenia-1, in the formation of synapses on cortical interneuron dendrites.     

Calmodulin Acts as an intermediary for the effects of calcium on gap junctions from crayfish lateral axons

Summary Lateral axons from the abdominal nerve cord of cray-fish were internally perfused with the calcium receptor calmodulin (CaM) in solutions with low (pCa>7.0) or high (pCa 5.5) calcium concentrations and studied electrophysiologically and morphologically. Results from these experiments show that when the internal solution contains calcium-activated calmodulin (Ca²⁺-CaM) the junctional resistance between the axons increases from control values of about 60 to 500–600 k in 60 min. In contrast, axons perfused with calmodulin in low calcium solutions maintain their junctional resistance at control levels during the 60-min perfusion. Similar results are obtained when only one or both coupled axons are perfused.The morphological study shows that in the perfused axons the axoplasmic organelles are replaced or grossly perturbed by the perfusion solution up to the region of the synapses. Additionally, in axons perfused with Ca²⁺-CaM there are regions where the synaptic gap between the membranes decreases from a control 4–6 to 2–3 nm. Both electrophysiological and morphological results can be interpreted as indicating that calcium-activated calmodulin acts directly on the junctional channels to induce their closure.     

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.     

The human thyroglobulin gene contains two 15-17 kb introns near its 3''-end.

We have cloned overlapping segments of the human thyroglobulin gene from a genomic cosmid library. Restriction mapping and electron microscopy show that a region of 38 kb at or near the 3'-end of this gene encodes only 850 nucleotides or 10% of the messenger RNA (mRNA) sequence. The region contains five exons of 130-210 nucleotides, split by introns of 1 to 15-17 kb. This represents the lowest ratio of coding to non-coding DNA (2.2%) found thus far in any eukaryotic gene. Blot hybridization under non-stringent conditions shows the presence of only one copy of this gene in the human genome and the absence of other closely related sequences.     

Molecular cloning, functional expression and pharmacological characterization of a human bradykinin B2 receptor gene.

The gene encoding a putative G protein-coupled receptor (HG10) was cloned from human genomic DNA by low stringency PCR and found to be homologous to the recently described rat bradykinin B2 receptor. The receptor was expressed in xenopus oocytes and stably transfected CHO cell lines. Binding studies demonstrated that HG10 encodes a high affinity BK receptor with an apparent Kd of 150 pM. Displacement by BK agonists and antagonists allowed the characterization of the receptor as a B2 subtype. Functional coupling to the Ca(2+)-phosphatidylinositol cascade was demonstrated in transfected CHO cells where inositol phosphates accumulation and intracellular calcium concentration were elevated in response to BK stimulation. The agonistic and antagonistic properties of BK analogs do not match strictly the pharmacological profile described for the rat or guinea pig B2 receptor subtypes or the putative B3 subtype. This discrepancy is attributed either to species variability or to differences in the coupling efficiency of receptors to the transduction cascade in different cell types. From our results, the existence of B3 receptors and of B2 subtypes appears questionable.     

BLUE-GREEN ALGAL MATS IN A SMALL STREAM1

Bedrock erosional features in a small stream (Little Schultz Creek, Bibb County, Alabama) created a variety of habitats for epilithic growth. One suck habitat was illustrated by the occurrence of small falls (<0.3 m) in the main channel of the stream and blue-green algal mats associated with them. The cohesive, laminar algal mats were found at 15 such sites along a 250-m reach of the stream. The primary mat matrix consisted of the blue-green alga Oscillatoria submembranacea Ardissone and Strafforella. The uppermost portion of each mat consisted of a thin (<1 mm thick) green layer of biologically active filaments. The lower layers were thicker (up to 2 cm thick) and consisted of brown laminae of Oscillatoria filaments, and associated sediments. In addition, numerous diatoms mere associated with the mat surface. Some were loosely attached (e.g. Achnanthes); others (Cymbella tumida (Bréb.) V. H.) were stalked. These mats were present throughout the year and showed a bimodal annual distribution with maxima hi February and July. In February, total mat coverage was higher than in July. This winter maximum may have been related to a mode of growth dependent upon sedimentation from storm events and subsequent upward growth of the alga. Mat primary productivity on an areal basis (432 mg C · m^?2· d^?1 in March and 907 mg C · M^?2· d^?1 in April) was 2–12 times the maxima measured on epizoic and cobbles surfaces and other bedrock surfaces in the same stream. The limited areal coverage of the mats, when compared to other surfaces available for algal colonization, made them less important than other epilithic and epizoic surfaces in terms of total primary production in this stream reach. However, we propose that the combination of their unique structure and high primary productivity may make these algal mats sites of high algal and bacterial metabolic activity, which may include anaerobic processes in midchannel, where such activity would not be expected to occur.     

Identification of hormonogenic domains in the carboxyl terminal region of bovine thyroglobulin

A segment of 986 nucleotides corresponding to the 3' end of the 8.5 kb bovine thyroglobulin (Tg) mRNA has been sequenced. An open reading frame of 302 codons was found, ending with TGA and preceding an 80 nucleotide long 3' untranslated sequence. The encoded protein sequence provided the first data on the carboxyl terminal portion of Tg. Lysine was identified as the last residue. Comparison of the amino acid sequence with that of peptides known to contain thyroid hormones in the mature protein, lead to the identification of three regions involved in thyroid hormone formation. Two closely linked thyroxine- forming sites were found 182 and 196 amino acids from the carboxyl terminus respectively. The antepenultimate amino acid of the protein corresponded to the recently described triiodothyronine-forming site. Together with the previous localization of the main thyroxine-containing peptide at the amino terminus, the present results provide a map of all hormonogenic sites identified to date in Tg.     

Restriction mapping of synthetic thyroglobulin structural gene as a means of investigating thyroglobulin structure

Bovine 33 S thyroglobulin mRNA was reverse transcribed into double-stranded DNA under conditions allowing the synthesis of a complete 8 kilobase pair copy. A physical map of the resulting synthetic thyroglobulin structural gene was constructed using six restriction endonucleases. The following conclusions could be drawn: (i) the polypeptide chains in thyroglobulin subunits are identical; (ii) thyroglobulin is composed of a major class of molecules sharing the same primary structure; (iii) there is no evidence for precise internal repetition in the structure of thyroglobulin subunits.