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R D Howells

Publications and source records attributed to R D Howells.

At least 55 records · Page 3Linked to original sources
PubMed1986

Cellular localization of proenkephalin mRNA in rat brain: gene expression in the caudate-putamen and cerebellar cortex.

The cellular locations of proenkephalin mRNA have been determined for the caudate-putamen and cerebellar cortex of the rat brain by in situ hybridization. In the caudate-putamen, more than half of the neurons express the proenkephalin gene. Morphologically, they are medium-sized cells that may represent projection neurons. In the cerebellar cortex, proenkephalin mRNA is present in a subpopulation of neurons in the granule layer that appear to be Golgi cells--i.e., inhibitory interneurons. The presence of [Met]enkephalin, a pentapeptide derived from proenkephalin, in these two brain areas is consistent with a synthetic role for this mRNA and implicates proenkephalin gene expression in the control of motor function.

Animals↗
PubMed1986

Proenkephalin mRNA in rat heart.

The distribution of preproenkephalin mRNA in rat tissues was investigated using a homologous cDNA probe for detection. The heart was found to contain larger amounts of the mRNA than any other tissue including brain, which heretofore had been considered the richest source. The identity of the message in heart was verified by hybridizing RNA blots with a synthetic oligodeoxynucleotide that recognizes a different region of the preproenkephalin mRNA sequence than does the cDNA probe. The preproenkephalin mRNA extracted from both heart and brain contained approximately equal to 1500 bases. Dissection of heart revealed that essentially all of the message is contained within the ventricles. In contrast to the large amounts of preproenkephalin mRNA in rat heart, the opioid peptide contents is only 3% of the amount in brain. The rat heart may be a useful model for the investigation of translation control of protein synthesis.

Animals↗
PubMed1986

IgG thyrotrophin receptor antibody activity in Graves' disease; a study of TSH agonist and antagonist activities by isoelectric focusing.

The distribution of TSH receptor antibody activity in the 7S and 19S fractions of Graves' sera has been re-evaluated. Serum fractions were obtained by gel filtration from 12 Graves' sera and assayed for TSH receptor binding activity in a radioreceptor assay. Thyroid stimulating activity was determined in a cultured porcine thyroid cell bioassay. In apparent contrast to the findings of Baker et al. (1983) TSH receptor binding activity was confined to the 7S gel filtration fraction, containing IgG, and was not detected in the 19S fraction, containing IgM. Similarly thyroid stimulating activity was detected only in the 7S fraction. 7S fractions from seven Graves' sera were fractionated by isoelectric focusing and the fractions analysed for TSH receptor binding activity and TSH agonist and antagonist activities. Five of the IgGs showed TSH agonist activity and in all five, the peak thyroid stimulating activity (measured by stimulation of cyclic AMP release from isolated porcine thyroid cells) was in fractions with a pI of between 8.0 and 9.5. In four of these five IgGs, TSH receptor binding activity showed similar isoelectric distribution to the thyroid stimulating activities. High levels of TSH receptor binding activity without associated TSH agonist or antagonist activity were however observed in some isoelectric fractions of the fifth stimulating Graves' IgG studied. All the isoelectric fractions from the fifth IgG with thyroid stimulating activities contained TSH receptor binding activity. Two of the Graves' IgGs showed TSH antagonist activity and both the TSH receptor binding and TSH antagonist activities of these IgGs showed similar isoelectric distribution with the peak activities at a pI of around 9.0. Consequently, it was not possible to separate TSH agonist or TSH antagonist activities from TSH receptor binding activity in seven Graves' sera by isoelectric focusing although in one IgG several isoelectric fractions contained isolated receptor binding activity. These findings are in keeping with the hypothesis that the biological activities of Graves' IgGs are intimately related to their ability to bind to the TSH receptor.

Animals↗
PubMed1985

Interaction of autoantibodies to thyrotropin receptor with a hydrophilic subunit of the thyrotropin receptor.

Reduction of human thyroid membranes with dithiothreitol caused the release of a water-soluble glycoprotein which neutralized the thyrotropin (TSH) receptor-binding and thyroid-stimulating activities of Graves' serum. Analysis of the protein by gel filtration and sucrose density gradient centrifugation allowed estimates of 3.45 nm for the Stokes' radius, 3.6 S for the s20,w and 47 000 +/- 5000 (mean +/- S.D.; n = 4) for the Mr. The material released by dithiothreitol treatment could be crosslinked to 125I-labelled TSH coupled to N-hydroxysuccinimidyl 4-azidobenzoate (125I-HSAB-TSH), suggesting that it contained a component of the TSH receptor. Furthermore, analysis of the crosslinked material by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis indicated that it contained the TSH receptor A subunit (Mr 50 000). Several factors suggested therefore that the glycoprotein released by dithiothreitol treatment of human thyroid membranes was the TSH receptor A subunit. In particular, (a) both preparations were hydrophilic and were released from membranes by reduction, (b) they had similar Mr values and (c) both preparations crosslinked to 125I-HSAB-TSH. Material similar to the TSH receptor A subunit was released from thyroid membranes by treatment with papain, probably as a result of cleavage of the receptor A subunit at a site close to the interchain disulphide bridge. A similar mechanism, involving thyroid proteinases, was probably involved in release of material with similar properties to the TSH receptor A subunit during freezing and thawing of human thyroid homogenates.

Autoantibodies↗
PubMed1985

Analysis of thyrotropin receptors by photoaffinity labelling. Orientation of receptor subunits in the cell membrane.

Porcine thyrotropin (TSH) receptors have been purified by Sepharose-TSH affinity chromatography and crosslinked to a 125I-labelled photoactive derivative (N-hydroxysuccinimidyl 4-azidobenzoate; HSAB) of TSH (125I-HSAB-TSH). Purification of the crosslinked complexes on Sephacryl S-300 followed by polyacrylamide-gel electrophoresis in sodium dodecyl sulphate showed that the receptor contained two subunits. One subunit (A) with Mr 45 000 was crosslinked to TSH and the other (B) subunit, Mr 25 000, was linked to the A subunit by a disulphide bridge(s). Other, as yet unidentified, subunits may have been non-covalently associated with the A and B subunits. Analysis of reduced and non-reduced crosslinked TSH receptor-125I-HSAB-TSH on Sephacryl S-300 in the presence and absence of detergent indicated that the A subunit was a hydrophilic peptide. This was confirmed in studies of the release into aqueous solution by reducing agent treatment of 125I-HSAB-TSH crosslinked to the TSH receptor A subunit in thyroid membranes. Similar results were obtained with TSH receptors in human thyroid and guinea pig fat cell membranes. These studies suggest that the hydrophilic A subunit of the receptor forms a binding site for TSH on the outside surface of the cell membrane and that the A subunit is linked to the cell membrane by way of a disulphide bridge to the receptor B subunit.

Affinity Labels↗
PubMed1985

Affinity-labelling of the thyrotropin receptor. Characterization of the photoactive ligand.

Thyrotropin (TSH) has been coupled to the photoactive heterobifunctional reagent N-hydroxysuccinimidyl 4-azidobenzoate (HSAB) and the properties of the product (HSAB-TSH) investigated. Preparations of HSAB-TSH containing two molecules of HSAB per molecule of TSH were used in most experiments and these preparations retained about 40% of the original receptor-binding activity of the TSH. HSAB-TSH could be labelled with 125I and cross-linked to porcine and human TSH receptors. Analysis of the cross-linked complexes indicated that the receptors consisted of two subunits (designated A and B) linked by a disulphide bridge. In the case of the human TSH receptor, the A- and B-subunits had approximate Mr values of 50 000 and 30 000 respectively, whereas the Mr values for porcine TSH-receptor A- and B-subunits were approx. 45 000 and 25 000 respectively. Only the A subunit was cross-linked to TSH. Comparison of the effects of trypsin and mercaptoethanol on the TSH-TSH-receptor complexes suggested that the trypsin cleavage point on the A-subunit was at a point close to the disulphide bridge.

Affinity Labels↗
PubMed1985

Expression of preproenkephalin-like mRNA and its peptide products in mammalian testis and ovary.

The distribution of preproenkephalin mRNA and proenkephalin-derived peptides have been examined in gonadal tissues from rats, hamsters, and cattle. A preproenkephalin mRNA band was detected in the ovaries of all three species and in hamster testis that is identical in size to the 1450-nucleotide mRNA typically found in tissues that express proenkephalin. Rat testis, on the other hand, expresses at least one preproenkephalin-like mRNA that is substantially greater in size (1900 nucleotides). [Met]enkephalin-containing peptides were also detected in each of the gonadal tissues examined. Although the abundance of preproenkephalin-like mRNA in rat testis was comparable to that in rat brain, the testicular content of proenkephalin-derived [Met]enkephalin sequences was less than 4% of the rat brain content. Together these data suggest that preproenkephalin-like mRNA in rat testis is not efficiently translated, proenkephalin-derived peptides undergo rapid turnover in this tissue, or the mRNA in rat testis has a frameshift resulting in an altered coding sequence.

Animals↗
PubMed1985

Immunoprecipitation of TSH-TSH receptor complexes.

The ability of Graves' sera to interact with the TSH receptor crosslinked to a 125I-labelled photoactive derivative of TSH has been investigated. Crosslinked complexes were prepared using non-purified detergent solubilized human thyroid and guinea pig fat TSH receptors. Affinity purified porcine TSH receptor preparations wee also used. After crosslinking, the crosslinked TSH-TSH receptor complexes were separated from aggregates and free TSH on Sephacryl S-300, incubated with test sera followed by immunoprecipitation using anti-IgG or Protein A. Using non-purified human TSH receptors crosslinked to TSH, a mean +/- SD of 12.1 +/- 4.9% of the crosslinked complex was immunoprecipitated with Graves' sera (n = 7) compared with 10.3 +/- 2.6% with Hashimoto sera (n = 6; P greater than 0.14) and 3.8 +/- 1.0% with normal sera (n = 6; P less than 0.004). These values were markedly reduced when TSH receptor preparations free of other thyroid autoantigens (guinea pig fat TSH receptors) were used. Under these conditions immunoprecipitation with Graves' sera (n = 24) was 1.6 +/- 1.3% compared with 0.8 +/- 0.6% for Hashimoto sera (n = 13) and 0.8 +/- 0.4% for normal sera (n = 12; P less than 0.003). In addition complexes formed between TSH and affinity purified porcine TSH receptors gave low immunoprecipitation values for Graves' (1.44 +/- 0.73%; n = 20) and Hashimoto sera (1.7 +/- 0.94; n = 11) which were not significantly different (P greater than 0.4). Overall, therefore, the effects of Graves' and Hashimoto sera were similar and the amounts of material immunoprecipitated were markedly reduced when TSH receptor preparations containing reduced amounts of other autoantigens were used. Consequently the Graves' sera did not appear to interact specifically with crosslinked TSH-TSH receptor complexes. However the Graves' sera studied did contain TSH receptor antibodies which could inhibit the binding of labelled TSH to TSH receptors in the preparations used and our results suggest that the binding of TSH and these antibodies to the receptor is mutually exclusive. There is considerable evidence that serum from patients with Graves' disease contains antibodies to the TSH receptor (Rees Smith, 1981). Several studies have suggested that binding of the receptor antibody and TSH to the TSH receptor is mutually exclusive (Manley et al., 1977; Petersen et al., 1977; Rickards et al., 1981) but recently the formation of termolecular complexes consisting of detergent solubilized receptors, labelled TSH and Graves' IgG has been reported (Konishi et al., 1982; De Bruin et al., 1984).(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1985

Thyrotropin receptor antibodies.

The thyrotropin (TSH) receptor is an integral membrane protein which contains 2 subunits linked by a disulphide bridge. The A subunit (mol. wt. 50,000) is water soluble and forms the binding site for TSH, whereas the B subunit (mol. wt. 30,000) penetrates the lipid bilayer and probably forms the site for interaction with adenylate cyclase. Autoantibodies to the TSH receptor are found in the sera of patients with Graves' disease. The antibodies bind to the same region of the receptor's A subunit as TSH and usually act as TSH agonists, causing hyperthyroidism. Occasionally, TSH receptor autoantibodies are found which can act as TSH antagonists. Isoelectric focusing and binding studies indicate that these antibodies also bind to the same region of the receptor A subunit as TSH.

Animals↗
PubMed1984

Denervation of rat adrenal glands markedly increases preproenkephalin mRNA.

The effect of denervation on the expression of rat adrenal proenkephalin has been examined. Following splanchnicectomy there was a several-fold increase in the steady-state levels of preproenkephalin mRNA, which became maximal after 24-48 hr (greater than 10-fold). These results indicate that the previously observed increase in rat adrenal enkephalin-containing peptides following denervation occurs entirely by a pretranslational mechanism. The increase in preproenkephalin mRNA was accompanied by a 50-75% decrease in rat adrenal poly(A)+ RNA. Neural input thus exerts a profound trophic influence on proenkephalin gene expression and RNA metabolism in rat adrenals.

Adrenal Glands↗
PubMed1984

Molecular cloning and sequence determination of rat preproenkephalin cDNA: sensitive probe for studying transcriptional changes in rat tissues.

A cDNA probe was prepared to investigate the regulation of proenkephalin biosynthesis in the rat. This was necessary because human and bovine proenkephalin cDNA were not sensitive enough for the accurate detection of preproenkephalin mRNA in tissues that contain low copy numbers of this message, such as the adrenal gland. The rat probe was prepared in the following manner. Preproenkephalin mRNA was enriched by sucrose gradient centrifugation of poly(A)-containing mRNA from rat brain and was used as a template for double-stranded cDNA synthesis. The resulting cDNA was inserted into the plasmid pBR322, and recombinant plasmids were used to transform Escherichia coli RR1 cells. A synthetic oligodeoxyribonucleotide (30 bases long) with a sequence that had previously been shown to be identical in bovine and human preproenkephalin cDNA was prepared to screen the clone bank. The plasmid with the longest cDNA insert (about 1200 bases) from the positive clones was isolated, and the sequence of the entire protein coding region was determined. Like the bovine and human gene products, rat preproenkephalin contains four [Met]enkephalin sequences and one copy each of [Leu]enkephalin, [Met]enkephalin-Arg6-Gly7-Leu8, and [Met]enkephalin-Arg6-Phe7. Rat preproenkephalin is 80% and 83% homologous to the bovine and human forms, respectively, at the nucleotide level and is 82% homologous to both species at the amino acid level. Rat preproenkephalin contains 269 amino acid residues, making it larger than the human (267 residues) and bovine (263 residues) precursors. The sensitivity for detection of rat preproenkephalin mRNA with the rat cDNA was several times greater than with the corresponding cDNAs from bovine and human sources.

Amino Acid Sequence↗
PubMed1984

Intact proenkephalin is the major enkephalin-containing peptide produced in rat adrenal glands after denervation.

We have shown previously that enkephalin-containing peptides of high molecular weight increase 10- to 15-fold 3 days after adrenal denervation and have suggested that much of this material may represent newly synthesized proenkephalin. In this paper we report that most of the material appearing after denervation is indeed intact proenkephalin. The putative proenkephalin was partially purified by gel filtration chromatography and HPLC. A molecular size of about 25 kDa was estimated by gel filtration. On HPLC, the putative proenkephalin was eluted at a much higher propanol concentration than the 18.2-kDa enkephalin-containing peptide isolated previously. Treatment of this putative proenkephalin with endoproteinase Lys-C showed it contain [Met]-enkephalin, [Leu]enkephalin, [Met]enkephalin-Arg6-Phe7, and [Met]enkephalin-Arg6-Gly7-Leu8 in the same ratios as are found in proenkephalin as deduced from sequencing of proenkephalin cDNA.

Adrenal Glands↗
PubMed1983

Evidence for a proenkephalin-like precursor in amphibian brain.

The mammalian proenkephalin-derived peptides [Leu]enkephalin, [Met]enkephalin, and [Met]enkephalin-Arg6-Phe7 were identified in acid extracts of the brain of Bufo marinus by using reversed-phase HPLC and specific radioimmunoassays. [Met]Enkephalin was the predominant opioid peptide present (270 pmol/g). In contrast, the octapeptide [Met]enkephalin-Arg6-Gly7-Leu8, which is also derived from mammalian proenkephalin, was not detected. The ratio of free [Met]enkephalin to [Met]enkephalin-Arg6-Phe7 was found to be 3.5 to 1, which is similar to that observed in mammalian proenkephalin-containing tissues. Together these data (i) indicate that amphibian brain contains a proenkephalin related to the mammalian precursor and (ii) establish the existence of enkephalins and proenkephalin-derived enkephalin-containing peptides in a submammalian species.

Animals↗
PubMed1982

Solubilization and characterization of active opiate binding sites from mammalian brain.

Active binding sites have been solubilized from cell membranes derived from mammalian brain. High affinity, stereospecific binding to soluble sites was demonstrable when membranes from rat brain, human frontal cortex and bovine corpus striatum were treated with digitonin or glycodeoxycholate, provided that the binding assay was conducted at 25 degrees C or below and in the presence of 50 to 100 mM NaCl. The yield of solubilized binding sites extracted from brain cell membranes was increased substantially (up to 43% yield from bovine striatum) when membranes were treated with detergent solutions containing 0.5 to 1.0 M NaCl. This effect was not observed when LiCl, KCl or (NH4)2SO4 were substituted for NaCl. Evidence for the solubility of the binding sites was provided by two criteria: nonsedimentation after 2 hr of centrifugation at 10(5) X g and an apparent molecular weight of 3 to 4 X 10(5) as determined by gel filtration.

Animals↗