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R D Howells

Publications and source records attributed to R D Howells.

At least 37 records · Page 2Linked to original sources
PubMed1991

The measurement of complement fixation by autoantibodies directed against thyroid membrane antigens.

This paper describes the use of sensitized sheep red blood cells for the detection and titration of complement fixation by autoantibodies directed against human thyroid membranes in the serum of patients with autoimmune thyroid disease. Patients with elevated circulating levels of TPO antibodies and diagnosed as having autoimmune hypothyroidism (including Hashimoto's disease) or autoimmune hyperthyroidism (Graves' disease) were studied. Complement fixation titres were highest in those patients with autoimmune hypothyroidism compared with the autoimmune hyperthyroid group. Serum samples obtained from a group of patients with thyroid neoplasia and from normal healthy volunteers were negative in this test. The TPO antibody activity when "corrected" for its CF potency suggests that the autoantibodies found in autoimmune hypothyroidism are potentially more destructive than those found in the non-destructive autoimmune thyroid diseases.

Analysis of Variance↗
PubMed1990

Differential regulation of proenkephalin gene expression by estrogen in the ventromedial hypothalamus of male and female rats: implications for the molecular basis of a sexually differentiated behavior.

The ventrolateral aspect of the ventromedial hypothalamic nucleus (VL-VM) contains many estrogen-concentrating neurons which mediate estrogen facilitation of reproductive behavior. Previous studies have shown that estrogen treatment increases proenkephalin (PE) gene expression in neurons of the VL-VM in ovariectomized female rats, and that enkephalin peptides may stimulate lordosis behavior. To determine whether there is a sex difference in steroid hormone regulation of PE gene expression we have examined the effects of estrogen and testosterone on PE mRNA levels in male rats. Slot blot hybridization analysis of RNA isolated from the ventromedial hypothalamus indicated that estrogen treatment increased PE mRNA levels in the VL-VM of ovariectomized female rats (2.2-fold), but had no measurable effect on PE mRNA levels in gonadectomized males. Testosterone treatment of gonadectomized males also had no effect on PE gene expression. To determine whether the sex difference in estrogen-inducibility of PE gene expression is due to the developmental effects of gonadal steroids, we have investigated the effect of estrogen on PE mRNA levels in the VL-VM of neonatally androgenized female rats. Unlike the genetic male, the androgenized females responded to estrogen treatment with a female-typical increase in PE mRNA levels (1.7-fold). Further, although the androgenized rats clearly exhibited signs of defeminization, they did exhibit estrogen-facilitated lordosis behavior when tested with manual stimulation. The PE mRNA induction in estrogen-treated androgenized rats correlated well with the lordosis scores obtained by manual stimulation testing. These results indicate that estrogen regulation of PE gene expression in the VL-VM is sexually differentiated and support the hypothesis that the enkephalinergic neurons of the VL-VM are involved in the regulation of female reproductive behavior.

Animals↗
PubMed1990

Estradiol induction of proenkephalin messenger RNA in hypothalamus: dose-response and relation to reproductive behavior in the female rat.

Hormone effects on proenkephalin (PE) mRNA allow an opportunity to compare a brain region-specific molecular change with a quantifiable behavior. Slot blots were used to measure PE mRNA levels in the ventromedial hypothalamus (VMN) and preoptic area (POA) as a function of the dose of estrogen administered to ovariectomized rats. Every rat used had been characterized for the ability to display lordosis behavior. Estradiol treatment led to a monotonic dose-dependent increase in PE mRNA level in VMN, while only a small effect was observed in POA at the higher estradiol doses. Lordosis behavior, assessed manually and in mating tests, also increased monotonically with estradiol dose. The data indicate that an apparent 'threshold' level of PE mRNA in VMN coincided wit display of behavior, and suggest further that high levels of PE mRNA, alone, are not sufficient for lordosis. While the exact relationship of the eventual product, Met-enkephalin, to female reproductive behavior remains to be determined, the parallel changes in PE mRNA level and behavior encourage further analysis.

Animals↗
PubMed1990

Tissue-specific splicing of pro-enkephalin mRNA.

A testis cDNA library derived from 4-6 week-old rats was screened using a 32P-labeled 435 base pair (bp) cDNA probe derived from exon 3 of rat brain proenkephalin (PE). Two positive clones were isolated from an initial screening of approximately 20,000 recombinant bacteriophage plaques. The longest insert (1,500 bp) was sequenced and was found to contain a portion of intron A at the 5'-terminus. Intron B was sliced as it is in the brain, therefore, the domain structure of the cDNA was intron A-exon 2-exon 3, reading in the 5'-3' direction. Since the translational initiator codon is located at the 5'-terminus of exon 2, the reading frame of PE is unaltered, however, within intron A are 4 upstream AUG codons which exist in a favorable context for initiation of translation. It is probable that translation of PE would be inhibited by the presence of these upstream initiator codons, which may explain the discrepancy between the levels of PE mRNA and opioid peptides in the testis. Hypophysectomy prior to the onset of puberty was found to drastically reduce the level of PE mRNA in the testis and epididymus, suggesting that pituitary factors affect the expression of the PE gene in these tissues, either directly or indirectly.

Animals↗
PubMed1990

Primary standardization for the ELISA of serum thyroperoxidase and thyroglobulin antibodies and their prevalence in a normal Welsh population.

This paper describes the use of ELISA techniques, together with university available primary standards, for the assay of the thyroid autoantibodies anti-thyroperoxidase (TPOAb) and anti-thyroglobulin (TgAb). The data obtained using ELISA compared well with that obtained by passive haemagglutination giving correlation coefficients of 0.79 and 0.83 respectively. TPOAb and TgAb concentrations were assayed in serum samples from 832 healthy individuals aged between 18 and 45 years and cut-off values for both antibodies have been defined (TPOAb less than 19.6kIU/l; TgAb less than 98kIU/l). Applying these cut-off values, some 10% of the population showed elevated anti-thyroid antibody activity and, when divided by sex, a majority of this group belonged to the female population.

Adolescent↗
PubMed1989

Estrogen regulation of proenkephalin gene expression in the ventromedial hypothalamus of the rat: temporal qualities and synergism with progesterone.

Estrogen has been shown to increase proenkephalin (PE) mRNA levels in neurons of the ventrolateral aspect of the ventromedial hypothalamic nucleus (VL-VM). In this series of experiments, we examined the temporal qualities of this induction by determining both the latency of the estrogen-induced elevation in PE mRNA levels and the rate at which the message levels decline following removal of estrogen. In addition we have examined the effects of progesterone on PE gene expression in the VL-VM of estrogen-primed rats. The latency of the estrogen-induced elevation in PE mRNA levels was found to be relatively short: PE mRNA levels were increased 2-fold within 1 h of estrogen replacement. Following estrogen removal the levels of PE mRNA declined rapidly. Progesterone treatment attenuated this decline, prolonging the estrogen-induced elevation of PE mRNA levels. These results suggest that estrogen rapidly increases PE mRNA levels through a mechanism that probably involves alterations in both the rate of appearance and the rate of degradation of the message. Together, the short latency of the estrogen-induced elevation and the rapid rate of decay following estrogen removal indicate that PE gene expression is highly sensitive to fluctuating estrogen levels. The effect of progesterone suggests that this enkephalinergic system may be regulated by both estrogen and progesterone during the estrous cycle.

Animals↗
PubMed1989

A primary standard for the ELISA of thyroglobulin and microsomal autoantibody IgG subclass associated activity.

A primary standard for the assay of thyroid autoantibody subclass distribution was prepared by removing all but one IgG subclass from a standard serum by negative affinity chromatography. Affinity columns were prepared using murine monoclonal antibodies showing restricted specificity for human IgG (clone TM10-non-IgG1; HP6019-non-IgG2; VC9-nonIgG3 and 1a1-non-IgG4). Aliquots of patient serum were chromatographed and the eluted fractions assayed for IgG subclass concentration and thyroid autoantibody activity by ELISA. Recoveries of the desired IgG subclasses were: TM10 26.7%, HP6019 77.3%, VC9 53.0% and 1a1 46.0%. Contamination with unwanted subclasses was usually less than 1% but there was some "breakthrough" of 3 and 4 with HP6019. The thyroid autoantibody distribution, corrected for recovery and dilution, was thyroglobulin autoantibody (TgAb) IgG1 61.2%, IgG2 37.7%, IgG3 2.7%, IgG4 2.9% and thyroid microsomal autoantibody (MicAb) IgG1 73.7%, IgG2 19.8% IgG3 3.6% IgG4 3.8%. Several other serum samples were also analyzed by this technique and also by subclass ELISA calibrated with this standard serum. Regression analysis of the data obtained by the two assay methods gave correlation coefficients of r = 0.88 for TgAb and r = 0.82 for MicAb.

Antibodies, Monoclonal↗
PubMed1988

Estrogen increases proenkephalin messenger ribonucleic acid levels in the ventromedial hypothalamus of the rat.

The effects of estrogen on proenkephalin (PE) gene expression were measured in neurons of the ventromedial hypothalamus. Slot blot hybridization analysis indicates that the levels of PE mRNA in the ventromedial hypothalamus of ovariectomized rats increase 3.1-fold after 2 weeks of estrogen replacement. In situ hybridization reveals that the estrogen-inducible enkephalinergic neurons are located in the ventrolateral aspect of the ventromedial nucleus, a subnucleus known to contain many estrogen-concentrating neurons. The increase in PE mRNA levels is due to both a 63% increase in the number of detectable PE mRNA-containing neurons and a 2.0-fold increase in the levels of PE mRNA per enkephalinergic neuron (1.63 x 2.0 = 3.3-fold overall induction). This estrogen-regulated enkephalinergic cell group may represent part of the neural network mediating estrogen's effects on reproductive behavior and/or other neuroendocrine processes.

Animals↗
PubMed1988

Glucocorticoid regulation of enkephalins in cultured rat adrenal medulla.

The effect of dexamethasone on enkephalin-containing (EC) peptide levels and preproenkephalin mRNA levels was determined in adrenal medullary explants (glands) from sham and hypophysectomized (hypox) rats. Culture for 4 days in serum-free medium without dexamethasone resulted in a 13- and 4-fold increase in EC peptide levels in sham and hypox glands, respectively. The addition of dexamethasone (10(-5) M) produced a 20- to 26-fold increase in EC peptides in sham and hypox glands. In serum free medium, hypox glands showed a concentration dependent increase in EC peptides with the ED50 for dexamethasone equal to 5.7 x 10(-7) M. Since the glucocorticoid antagonist RU486 partially blocked the rise in EC peptides in sham glands, it appears that the increase in EC peptides in sham glands in the absence of dexamethasone is a result of a higher concentration of endogenous corticosterone in sham compared to hypox glands. Dexamethasone resulted in a 6-fold increase in preproenkephalin mRNA in hypox glands cultured for 2 days. This increase was approximately proportional to the increase in EC peptides seen at 4 days. In serum free medium progesterone, testosterone, and deoxycorticosterone failed to increase EC peptides in hypox glands. These results indicate that glucocorticoid treatment is required for maximal proenkephalin gene expression and EC peptide biosynthesis in cultured glands.

Adrenal Medulla↗
PubMed1987

Photoaffinity labelling of the TSH receptor on FRTL5 cells.

An investigation of the properties of TSH receptors on FRTL5 cells using affinity labelling with a 125I-labelled photoactive derivative of TSH is described. Our studies suggest that FRTL5 cells contain 2 principal types of cell surface TSH receptors. One form, probably a precursor, consists of a single polypeptide chain (Mr 120,000) with an intrachain loop of amino acids formed by a disulphide bridge. The other type of receptor consists of a water-soluble A chain (Mr 55,000) linked to an amphiphilic B chain (Mr 35,000) by a disulphide bridge. The 2 chain structure is probably derived from the single chain 120,000 protein by enzymatic cleavage of peptide sequences within the loop of amino acids formed by the intrachain disulphide bridge.

Affinity Labels↗
PubMed1987

Localization of preproenkephalin mRNA in the rat brain and spinal cord by in situ hybridization.

To determine the localization in rat brain and spinal cord of individual neurons that contain the messenger RNA coding for the opioid peptide precursor preproenkephalin, we performed in situ hybridization with a tritiated cDNA probe complementary to a protion of preproenkephalin mRNA. We observed autoradiographic signal over the cytoplasm of neurons of many regions of the central nervous system. Several types of controls indicated specificity of the labeling. Neurons containing preproenkephalin mRNA were found in the piriform cortex, ventral tenia tecta, several regions of the neocortex, nucleus accumbens, olfactory tubercle, caudate-putamen, lateral septum, bed nucleus of the stria terminalis, diagonal band of Broca, preoptic area, amygdala (especially central nucleus, with fewer labeled neurons in all other nuclei), hippocampal formation, anterior hypothalamic nucleus, perifornical region, lateral hypothalamus, paraventricular nucleus, dorsomedial and ventromedial hypothalamic nuclei, arcuate nucleus, dorsal and ventral premamillary nuclei, medial mamillary nucleus, lateral geniculate nucleus, zona incerta, periaqueductal gray, midbrain reticular formation, ventral tegmental area of Tsai, inferior colliculus, dorsal and ventral tegmental nuclei of Gudden, dorsal and ventral parabrachial nuclei, pontine and medullary reticular formation, several portions of the raphe nuclei, nucleus of the solitary tract, nucleus of the spinal trigeminal tract (especially substantia gelatinosa), ventral and dorsal cochlear nuclei, medial and spinal vestibular nuclei, cuneate and external cuneate nuclei, gracile nucleus, superior olive, nucleus of the trapezoid body, some deep cerebellar nuclei, Golgi neurons in the cerebellum, and most laminae of the spinal cord. In most of these brain regions, the present results indicate that many more neurons contain preproenkephalin mRNA than have been appreciated previously on the basis of immunocytochemistry.

Animals↗
PubMed1987

Haloperidol increases proenkephalin mRNA levels in the caudate-putamen of the rat: a quantitative study at the cellular level using in situ hybridization.

Previous immunocytochemical studies have shown that the opioid peptides, Met-enkephalin and Leu-enkephalin, are present in medium-sized, spiny projection neurons of the caudate-putamen. It has also been demonstrated that chronic treatment of rats with the dopamine receptor blocker, haloperidol, results in an increase in the levels of enkephalin peptides and proenkephalin mRNA in this brain region. To determine whether this increase in proenkephalin mRNA content is exhibited by all enkephalinergic neurons of the caudate-putamen or by only a subpopulation, we have used in situ nucleic acid hybridization to examine the haloperidol-induced increase in proenkephalin mRNA levels at the cellular level. Results of in situ hybridization suggest that all enkephalinergic neurons in the caudate-putamen can respond to haloperidol treatment with an increase in steady state levels of proenkephalin mRNA, and that the mean induction is an approximate 3-fold increase in the message levels. This suggests that dopamine exerts a tonic inhibitory effect on the expression of the proenkephalin gene in all of the enkephalinergic neurons of the caudate-putamen. Dot blot analysis indicated a 2.4-fold increase in the tissue levels of this mRNA. The agreement between the in situ hybridization results and dot blot analysis supports in situ hybridization as a reliable method for quantitative studies of alterations in neuropeptide precursor mRNAs in the brain.

Animals↗
PubMed1987

Structure-activity analysis of microsomal antigen/thyroid peroxidase.

The interaction between thyroid microsomal autoantibodies and thyroid microsomal antigen/thyroid peroxidase (TPO) has been studied using both intact antigen preparations and their water-soluble trypsin fragments. In an analysis of sera from 30 patients with Graves' or Hashimoto's diseases, microsomal antibodies showed similar reactivity towards trypsin fragments (with TPO activity) and intact detergent (sodium deoxycholate, DOC)-solubilized human microsomal antigen preparations (r = 0.96). This raised the possibility that both the peroxidase-active site and the major autoantigenic site(s) of microsomal antigen were present on the same trypsin fragments. Studies with porcine TPO showed that only a few sera contained microsomal antibodies which cross-reacted strongly with the porcine preparations. Further analysis was carried out by immunoprecipitation of 125I-labelled microsomal antigen followed by SDS-PAGE and autoradiography. These studies suggest that intact human microsomal antigen (a single-chain protein with Mr = 110,000) contains an intrachain loop of amino acids formed by a disulphide bridge. Trypsin treatment cleaves the antigen close to its transmembrane section and releases a water-soluble fragment (Mr = 100,000), containing the intact disulphide-linked loop of amino acids. Further trypsin action causes cleavage of the peptide bonds within the loop in some preparations. Consequently, three major water-soluble trypsin fragments (Mr = 100,000, 73,000 and 68,000) are formed all of which contain an intact disulphide bridge and have microsomal antibody binding activities. The integrity of the disulphide bridge in intact antigen/TPO preparations and their trypsin fragments is essential for autoantibody binding activity.

Animals↗
PubMed1987

The TSH receptor: structure and interaction with autoantibodies in thyroid disease.

Studies of the TSH receptor using affinity labelling with photoactive derivatives of TSH and analysis by SDS-PAGE have shown that the receptor contains 2 subunits (A and B), linked by a disulphide bridge. Similar results are obtained with TSH receptors from human, porcine and guinea pig thyroid tissue and from guinea pig fat. Analysis of affinity labelled receptors under non-denaturing conditions suggest that subunits additional to the A and B subunits are not present. Hydrodynamic measurements indicate that the receptor A subunit has an approximately spherical structure (Stokes' radius 70 A) and when this interacts with TSH (an elongated structure with Stokes' radius 56A) a very elongated complex (Stokes' radius 104A) is formed. Isoelectric focusing studies of the TSH receptor A subunit, TSH and TSH receptor antibodies indicate that charge-charge interactions are of considerable importance in the binding of hormone and antibody to the receptor.

Animals↗
PubMed1986

Thyrotropin cross-links to the thyrotropin receptor through both the alpha and beta subunits.

We have recently shown that the beta subunit of thyrotropin (TSH) can be cross-linked to the TSH receptor [Buckland, Strickland, Pierce & Rees Smith (1985) Endocrinology (Baltimore) 116, 2122-2124; Buckland, Strickland & Rees Smith (1985) Biochem. Soc. Trans. 13, 942-943]. We failed, however, to cross-link the alpha subunit to the receptor, leaving the role of this subunit in the TSH-TSH-receptor interaction uncertain. We now report the successful cross-linking of the TSH alpha subunit to the receptor by the use of two different cross-linking reagents. Our studies suggest therefore that both subunits of TSH form part of the hormone's receptor-binding site.

Azides↗
PubMed1986

Isoelectric focusing of the human TSH receptor A subunit.

The water soluble A subunit of the human TSH receptor has been shown to have an isoelectric point of 5. As both TSH and TSH receptor antibodies have isoelectric points in the region of 8-10, charge-charge interactions must be of major importance in the binding of hormone or antibody to the TSH receptor A subunit.

Humans↗
PubMed1986

Assessment of the shape and molecular size of TSH-TSH receptor complexes.

Photoaffinity labelling and analysis under denaturing conditions (SDS-PAGE) have shown that the porcine TSH receptor contains an A subunit (Mr = 47,000) which forms the binding site for TSH and a B subunit (Mr = 25,000) linked to the A subunit by a disulphide bridge. In order to assess the size and shape of the receptor under non-denaturing conditions we have solubilized photoaffinity-labelled porcine TSH receptors using the small micelle-sized detergent sodium deoxycholate and analysed the preparations by sucrose density gradient centrifugation and gel filtration. Under these conditions, the cross-linked TSH-TSH receptor complex showed an S20,w of 6.4 S and a frictional ratio f/f0 of 1.8. These values were consistent with those which might be expected from an elongated protein complex with a molecular weight of about 100,000 (the value obtained by SDS-PAGE). Analysis of another thyroid membrane protein, human thyroid microsomal antigen (Mr = 110,000 by SDS-PAGE) under the same conditions gave an S20,w of 6.0 S and f/f0 = 1.3, suggesting that this protein has a compact structure. The TSH receptor A subunit cross-linked to TSH (Mr = 70,000 by SDS-PAGE) gave an S20,w of 4.6 S and f/f0 = 1.8 and these values could be compared with those obtained for the A subunit alone (S20,w = 3.6 S; f/f0 = 1.4; Mr by SDS-PAGE = 47,000) and TSH alone (S20,w = 2.6 S; f/f0 = 1.6; Mr = 28,000.(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity Labels↗