Secretion of soluble functional insulin receptors by transfected NIH3T3 cells

In order to determine whether the human insulin receptor ectodomain can be expressed as a functional protein, the coding regions for the transmembrane and cytoplasmic domain of a full-length human insulin receptor cDNA were deleted by site-directed mutagenesis, and the resultant construct was inserted into a bovine papilloma virus vector under the control of the mouse metallothionein promoter. After transfection of mouse NIH3T3 cells, a cell line secreting an insulin binding protein was isolated. The insulin binding alpha subunit had an Mr of 138,000 and a beta subunit of Mr 48,000 (compared to 147,000 and 105,000 for the full-length human insulin receptor expressed in NIH3T3 cells). This difference in size of the alpha subunit was due to a difference in glycosylation as N-glycanase digestion reduced the apparent size of the alpha subunits of secreted and normal membrane-bound receptors to identical values. The secreted receptor formed disulfide-linked heterotetrameric structures with an Mr of 280,000. It was synthesized as an Mr 160,000 precursor which was cleaved into mature subunits with a t1/2 of 3 h. Increasing expression of the cDNA by induction with sodium butyrate lead to the appearance of an Mr 180,000 protein in the medium as well as the mature alpha and beta subunits. A Scatchard plot of insulin binding to the secreted receptor was curvilinear with a Kd of 7 X 10(-10) M for the high affinity sites and 10(-7) M for the low affinity site (compared to Kd values of 1.1 X 10(-9) M and 10(-7) M, respectively, for human insulin receptors expressed in these cells.     

Inhibition of RNA synthesis in embryo of Xenopus laevis by protease inhibitor

We have studied the role of proteases during the development of Xenopus laevis embryos with the aid of protease inhibitors. The activity of proteases was found to be only minimal in the unfertilized egg and during the initiation of development, but activity began to increase at the morula stage. When the activity of proteases was inhibited by antipain, an inhibitor of endopeptidase activity, RNA synthesis in the embryo was inhibited. To examine the relationship between the inhibitory effect of antipain on protease activity and its effect on RNA synthesis, antipain was reduced with NaBH4 to inactivate its protease inhibitory activity. The reduced antipain did not inhibit RNA synthesis in the embryo. Antipain effectively inhibited synthesis of both rRNA and poly(A)+RNA but not 4S RNA. We therefore suggest that protease activity plays an important role in the initiation and/or continuation of RNA synthesis.     

Point mutations in the upstream region of the α-galactosidase A gene exon 6 in an atypical variant of Fabry disease

Summary Single point mutations in the upstream region of exon 6 of the -galactosidase A gene were found in two Japanese cases of the cardiac form of Fabry disease; ³⁰¹ArgGln (⁹⁰²GA) in a case that has already been published and ²⁷⁹GlnGlu (⁸³⁵CG) in a new case. They both expressed markedly low, but significant, amounts of residual activity in COS-1 cells. In contrast, two unrelated cases with classic Fabry disease were found to have different point mutations, which showed a complete loss of enzyme activity in a transient expression assay; ³²⁸GlyArg (⁹⁸²GA) in the downstream region of exon 6 in one case and two combined mutations, ⁶⁶GluGln (¹⁹⁶GC)/¹¹²ArgCys (³³⁴CT), in exon 2 in the other. We conclude, on the basis of the results recorded in this study and those in previous reports, that the pathogenesis of atypical Fabry disease is closely associated with point mutations in the upstream region of exon 6 of the -galactosidase A gene.     

Innervation of the C cells of chicken ultimobranchial glands studied by immunohistochemistry, fluorescence microscopy, and electron microscopy

Innervation of the ultimobranchial glands in the chicken was investigated by immunohistochemistry, fluorescence microscopy and electron microscopy. The nerve fibers distributed in ultimobranchial glands were clearly visualized by immunoperoxidase staining with antiserum to neurofilament triplet proteins (200K-, 150K- and 68K-dalton) extracted from chicken peripheral nerves. The ultimobranchial glands received numerous nerve fibers originating from both the recurrent laryngeal nerves and direct vagal branches. The left and right sides of the ultimobranchial region were asymmetrical. The left ultimobranchial gland had intimate contact with the vagus nerve trunk, especially with the distal vagal ganglion, but was somewhat separated from the recurrent nerve. The right gland touched the recurrent nerve, the medial edge being frequently penetrated by the nerve, but the gland was separated from the vagal trunk. The left gland was innervated mainly by the branches from the distal vagal ganglion, whereas the right gland received mostly the branches from the recurrent nerve. The carotid body was located cranially near to the ultimobranchial gland. Large nerve bundles in the ultimobranchial gland ran toward and entered into the carotid body. By fluorescence microscopy, nerve fibers in ultimobranchial glands were observed associated with blood vessels. Only a few fluorescent nerve fibers were present in close proximity to C cell groups; the C cells of ultimobranchial glands may receive very few adrenergic sympathetic fibers. By electron microscopy, numerous axons ensheathed with Schwann cell cytoplasm were in close contact with the surfaces of C cells. In addition, naked axons regarded as axon terminals or "en passant" synapses came into direct contact with C cells. The morphology of these axon terminals and synaptic endings suggest that ultimobranchial C cells of chickens are supplied mainly with cholinergic efferent type fibers. In the region where large nerve bundles and complex ramifications of nerve fibers were present, Schwann cell perikarya investing the axons were closely juxtaposed with C cells; long cytoplasmic processes of Schwann cells encompassed large portions of the cell surface. All of these features suggest that C-cell activity, i.e., secretion of hormones and catecholamines, may be regulated by nerve stimuli.     

Effect of substitution of glycine for arginine at position 146 of the A1 subunit on biological activity of Escherichia coli heat-labile enterotoxin.

The ADP-ribosyltransferase activity of polypeptide A1 of cholera toxin and that of Escherichia coli heat-labile enterotoxin (LT) are primarily responsible for the toxic activities of these toxins. Since the amino acid sequences of the two A1 polypeptides are very similar, their functional mechanisms are considered to be the same. Arg-146 of polypeptide A1 is thought to be involved in the active site, because this amino acid of cholera toxin has been identified as the site of self-ADP-ribosylation. However, the exact role of Arg-146 and the significance of self-ADP-ribosylation in toxicity remain unclear. We substituted Arg-146 of polypeptide A1 of LT with Gly by oligonucleotide-directed mutagenesis and examined the biological property of the resultant mutant LT. The substitution changed the mobility of subunit A on sodium dodecyl sulfate-polyacrylamide gel but did not reduce the vascular permeability activity of LT. This result indicates that Arg-146 is not absolutely required for toxic activity and that LT can express its toxic activity without self-ADP-ribosylation at Arg-146.