Thyroid hormone down-regulates p55, a thyroid hormone-binding protein that is homologous to protein disulfide isomerase and the beta-subunit of prolyl-4-hydroxylase

We have previously characterized a cellular thyroid hormone-binding protein (p55) that is found concentrated on the lumenal face of the endoplasmic reticulum and nuclear envelope (Cheng, S.-y., Hasumura, S., Willingham, M.C., and Pastan, I. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 947-951). To understand the role p55 plays in thyroid hormone action, we examined the regulation of p55 by 3,3',5-triiodo-L-thyronine (T3). Rat pituitary tumor GH3 cells cultured in regular medium, thyroid hormone-depleted medium (Td medium), or Td medium supplemented with 50 nM T3 (Td + T3 medium) were metabolically labeled with [35S]methionine and immunoprecipitated with antibodies against p55. Treatment with T3 caused a fall in p55 levels. Poly(A+) RNA from cells cultured in regular, Td, or Td + T3 medium was hybridized to a cDNA from p55. T3 withdrawal or addition had no effect on p55 mRNA levels. Furthermore, the initial rates of synthesis of p55 from cells cultured in regular, Td, and Td + T3 were found to be similar. However, analysis of the decay curves from cells in which p55 was pulse-labeled with [35S]methionine indicated that p55 is 2-fold less stable in T3 containing medium. These results indicated that down-regulation of p55 by T3 occurs at the post-translational level. Since DNA sequence analysis indicates that p55 is identical to protein disulfide isomerase and the beta-subunit of prolyl-4-hydroxylase, T3 may mediate its effects on the synthesis, secretion, and/or transport of proteins via p55.     

Characterization and chemical properties of phosphoribosylamine, an unstable intermediate in the de novo purine biosynthetic pathway

Incubation of [1-13C]-5-phosphoribosyl pyrophosphate ([1-13C]PRPP) and glutamine with PRPP amidotransferase results in rapid production and disappearance of two new resonances at 89.3 and 85.9 ppm. These resonances coincide with two of the products produced upon incubation of [1-13C]ribose 5-phosphate with NH3. Extensive NMR studies (15N and 1H-13C chemical shift correlation spectra) have allowed assignment of these resonances to beta- and alpha-phosphoribosylamine. These studies represent the first spectral observations of this chemically reactive intermediate. The rate of interconversion of alpha- to beta-phosphoribosylamine as a function of pH has been determined by saturation and inversion-transfer NMR methods. The rate of formation of 5-phosphoribosylamine (PRA) from ribose 5-phosphate and NH3 and its rate of decomposition as a function of pH have been determined with a glycinamide ribonucleotide synthetase trapping system fashioned after earlier studies of Nierlich and Magasanik [Nierlich, D. P., & Magasanik, B. (1965) J. Biol. Chem. 240, 366]. Phosphoribosylamine has a t1/2 = 38 s at 37 degrees C and pH 7.5. The pH-independent equilibrium constant for ribose 5-phosphate and NH3 with phosphoribosylamine has been established, 2.5 M-1, by use of these rate constants as well as by NMR methods. This equilibrium constant and the rates of nonenzymatic interconversion of alpha- and beta-PRA provide essential background for studying the mechanism of glycinamide ribonucleotide synthetase and investigating the possibility of channeling phosphoribosylamine between this enzyme and the first enzyme in the purine pathway.     

Synthesis and secretion of mucous glycoprotein by the gill of Mytilus edulis. I. Histochemical and chromatographic analysis of [14C]glucosamine bioincorporation

The ability of the isolated gill epithelium of Mytilus edulis to incorporate [14C]glucosamine as a precursor in the biosynthesis and secretion of mucous glycoproteins was investigated. Localization of mucous cells in the gill filament was achieved using histochemical staining techniques. Mucus cells containing neutral and acidic mucins were found in the lateral region, whereas mucus cells containing primarily neutral or sulfated mucins were found in the abfrontal region. Autoradiographic results showed that in both regions, the mucous cells were rich in content of the incorporated radiolabel. The secreted glycoproteins containing the incorporated radiolabel were analyzed by column chromatography using Bio-Gel P-2 and P-6. Two populations of the glycoproteins differing in molecular size were isolated. Upon alkaline reductive borohydride cleavage of the O-glycosidic linkages of the high molecular weight protein, about 70% of the radiolabel and 85% of the carbohydrate content were removed from the protein. The alkaline borohydride cleavage resulted in the formation of at least six oligosaccharide chains of various lengths of sugar units. Gas chromatographic analysis of the carbohydrate composition shows that the glycoproteins contain N-acetylglucosamine, N-acetylgalactosamine, and galactose, fucose, and mannose as the neutral monosaccharides. The above results indicate that the isolated gill epithelium of M. edulis is capable of incorporating [14C]glucosamine in the synthesis of secretable mucin-type glycoproteins.     

Membrane-buffer partition coefficients of tetracaine for liquid-crystal and solid-gel membranes estimated by direct ultraviolet spectrophotometry

The membrane-buffer partition coefficient of tetracaine was measured by direct ultraviolet spectrophotometry in dimyristoylphosphatidylcholine unilamellar liposomes at temperatures above and below the main phase transition. The partition coefficients of uncharged tetracaine to solid-gel (18 degrees C) and liquid-crystal (30 degrees C) membranes were 6.9 x 10(4) and 1.2 x 10(5), respectively. Despite the general assumption that local anesthetic binding to the solid membrane is negligible, this study showed that the solid membrane binding amounts to 57.5% of the liquid membrane binding. Binding of the charged form to the liquid or solid membrane was not detectable under the present experimental condition of 0.03 mM tetracaine bulk concentration. The present method measures metachromasia of local anesthetics when bound to lipid membranes. Its advantage is that the separation of the vesicles from the solution is not required. A linearized equation is presented that estimates the partition coefficient or binding constant graphically from a linear plot of the absorbance data. The method is applicable for estimation of drug partition when a measurable spectral change occurs due to complex formation.     

Variation in crypt size and its influence on the analysis of epithelial cell proliferation in the intestinal crypt.

The standard model of epithelial cell renewal in the intestine proposes a gradual transition between the region of the crypt containing actively proliferating cells and that containing solely terminally differentiating cells (Cairnie, Lamerton and Steel, 1965 a, b). The experimental justification for this conclusion was the gradual decrease towards the crypt top of the measured labeling and mitotic indices. Recently, however, we have proposed that intestinal crypts normally undergo a replicative cycle so that at any time in any region of the intestine, crypts will be found to have a wide range of sizes. We show here that if this intrinsic size variation is taken into account, then a sharp transition between the proliferative and nonproliferative compartments of individual intestinal crypts is consistent with the labeling and mitotic index distributions of mouse and rat jejunal crypts. Thus there is no need to invoke the region of gradual transition from proliferating to nonproliferating cells as is done in the standard model. The position of this sharp transition is estimated for both the mouse and rat. Experiments to further test our model are suggested and the significance of the results discussed.     

Absence of increased oxygen consumption in brown adipose tissue of rats exhibiting "cafeteria" diet-induced thermogenesis

Young male Sprague-Dawley rats were induced to overeat (approximately 45%) by provision of a "cafeteria" (CAF) diet of palatable human foods. Normophagic rats fed a commercial chow or a semisynthetic diet served as controls. The CAF rats exhibited (a) the reduced food efficiency and the propranolol-inhibitable elevation in resting metabolic rate (resting VO2) that are indicative of a facultative diet-induced thermogenesis (DIT) by which excess energy gain is resisted, and (b) certain changes in brown adipose tissue (BAT) that are among those taken as evidence for BAT as the effector of DIT, e.g., increased protein content and increased mitochondrial binding of GDP. To assess directly and quantitatively the contribution by BAT to the elevation in VO2 (apparent DIT) of the CAF rats, BAT O2 consumption was determined (Fick principle) from measurements of tissue blood flow (microsphere method) and the arteriovenous difference in blood O2 across interscapular BAT (IBAT). To obtain the measurements, the animals were fitted under halothane anesthesia with vascular cannulas for intraventricular injection of microspheres and sampling of arterial blood and the venous effluent of IBAT. After recovery from anesthesia and rewarming to normal body temperature the animals were placed singly in a temperature-controlled metabolic chamber and the measurements, which also included determination of resting VO2, were made 1.5-2 h later about 11:30 h. As determined from measurements made at 28 degrees C (thermoneutrality) mean values of resting VO2 for the cannulated rats were unchanged from those of intact (unoperated) CAF or control rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis of epidermal growth factor (EGF) receptor in vitro using SP6 RNA polymerase-transcribed template mRNA

The epidermal growth factor (EGF) receptor plays a key role in the control cellular proliferation, and its homology to the avian erythroblastosis virus erb B oncogene implicates its involvement in cellular transformation. The establishment of a correlation between the various structural domains of the EGF receptor and their functional counterparts would greatly advance our understanding of these processes. To this end, we have constructed an expression vector containing the SP6 viral promoter and an adjacent cDNA fragment encoding the full-length EGF receptor. Upon addition of SP6 RNA polymerase, this DNA is capable of generating large amounts of EGF receptor mRNA; this RNA can then be translated in vitro into immunoprecipitable EGF receptor protein. The translational efficiency of this EGF receptor RNA was found to be relatively low: approx. 100-fold lower than globin RNA synthesized using SP6 RNA polymerase. Use of these tools should now permit the synthesis and analysis of mutated EGF receptor protein in an effort to clarify the role of this receptor in growth control.     

The noradrenaline content and innervation of brown adipose tissue in the young rabbit

This work examined the noradrenaline content of brown adipose tissue, the metabolic response to endogenous noradrenaline released during tyramine infusion, and the innervation of brown fat at the electron microscopic level in the young rabbit. The noradrenaline content (ng/g) of the interscapular and cervical fat deposits ranged from 256 +/- 51 to 343 +/- 59 and 399 +/- 18 to 694 +/- 92, respectively, in four groups of rabbits (1-2, 7-8, 12-13, and 25-27 days of age). There was considerable variation amongst animals in each age group, but no evidence of a major increase or decrease in noradrenaline content during the first 4 weeks of life. Intravenous infusion of tyramine (100 micrograms X kg-1 X min-1) increased plasma noradrenaline concentration, oxygen consumption, and blood flow to brown fat. Thus noradrenaline released from endogenous sites, as well as injected noradrenaline, will initiate the thermogenic response of brown fat. Ultrastructurally, unmyelinated axons that were not organized in a fascicle were observed adjacent to the adipocytes in the late gestation fetus. By 1 week of age of axons were surrounded by Schwann cell cytoplasm which formed a fascicle. However, no evidence of myelination was found up to 21 days of age. Collectively, the data indicate that the brown adipocyte is fully responsive at 1-2 days of age even though myelination of the nerves is incomplete, and that the incomplete development of the sympathetic nerves at birth is not a factor in the synthesis of noradrenaline in the very young rabbit. In addition, brown fat of the newborn rabbit is not as thermogenically active as the brown fat of the cold-acclimated rat.     

The stereochemical course of phosphoryl transfer catalyzed by herpes simplex virus type I-induced thymidine kinase

Herpes simplex virus type I (HSV-I)-induced thymidine kinase has been shown to catalyze phosphoryl transfer from adenosine 5'-[gamma-(S)-16O,17O,18O]triphosphate to thymidine with inversion of configuration at phosphorus. The simplest interpretation of this result is that phosphoryl transfer occurs by a single in-line group transfer between ATP and thymidine within the ternary enzyme complex.