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Biomedical subjects

N Hunt

Publications and source records attributed to N Hunt.

At least 91 records · Page 5Linked to original sources
PubMed1993

Serum unmasks the binding of thyroid-stimulating hormone to endogenous and transfected receptors: evidence for a soluble form of the receptor in human thyroid.

A specific homologous radioligand receptor assay for thyroid-stimulating hormone (TSH) using bovine thyroid membranes was adapted for use with human thyroid. Specific 125I-labelled TSH binding was detected in the 3000 g membrane pellet from bovine thyroid but predominantly in the 3000 g supernatant of the human thyroid homogenate. Both assays required incubation in the presence of 10% serum, whilst the assay using human thyroid could only be precipitated using polyethylene glycol (PEG). The serum requirement transcended a possible role as carrier protein and unmasked specific TSH binding. Molecular sieving determined that the active fraction of the serum had an apparent size of 30,000-100,000. The requirement for PEG-assisted precipitation of the TSH receptor assay was a consequence of the TSH-binding entity from Graves' thyroid behaving like a soluble 'receptor': it did not sediment with the membranes, passed a 0.2 microns filter and, upon molecular sieving, had an apparent size of 300,000-1,000,000. A full-length TSH receptor cDNA was cloned from a human Graves' thyroid library and stably transfected cell lines expressing the TSH-receptor protein were constructed using human HeLa and murine 3T3 cells. Specific TSH binding was unmasked by serum in the human cell lines, as observed for the human thyroid TSH receptor, whereas serum hindered TSH binding in the murine cell lines. A soluble form of the receptor was not released from the cells and was not produced in conditions which demonstrated a soluble receptor-like binding component in human thyroid tissue.

Animals↗
PubMed1993

Expression of recombinant human thyroid peroxidase by the baculovirus system and its use in ELISA screening for diagnosis of autoimmune thyroid disease.

The cDNAs coding for human full-length and soluble thyroid peroxidase (TPO) were constructed, cloned into a baculovirus transfer vector and used for infection of Spodoptera frugiperda (Sf9) cells. The soluble TPO lacking 87 amino acids of the C-terminal transmembrane and intracisternal domains was designed as a fusion protein with a histidine-hexapeptide as an affinity ligand at its C-terminus. Whereas the recombinant full-length TPO was expressed mainly in an insoluble form in Sf9 cells, the recombinant soluble TPO was almost completely secreted into the culture medium. Both the full-length and the soluble TPO were purified by conventional methods and by a specific affinity chromatography using metal chelating matrix respectively, and tested for their autoantigenicity towards anti-TPO autoantibodies. The ELISA established with the purified recombinant soluble TPO as antigen demonstrated its specificity, practicability and reproducibility in screening of anti-TPO autoantibodies in sera of autoimmune thyroid patients. High correlation (r = 0.89, n = 175) was obtained between the soluble TPO and natural TPO prepared from human thyroid glands. Pathological sera (n = 200) were positively assayed with a significance of 91%.

Animals↗
PubMed1992

Life events and relapse in bipolar affective disorder.

In a 2 year study of life events and relapse in a cohort of 62 patients with bipolar affective disorder, an excess of events was found during the month immediately preceding relapse. Of 52 relapses 19% were preceded by a severe event in the previous month, compared to a background rate of 5% of patients experiencing a severe event each month at other times. The rate of life events prior to relapse was not apparently different between manic and depressive relapse, though the number of depressive episodes was small.

Adult↗
PubMed1992

Vasopressin biosynthesis in rodent Leydig cells.

Local biosynthesis of the peptide hormone vasopressin is demonstrated in vitro in Leydig cells derived from rat and mouse testis. Cycloheximide-sensitive production of the nonapeptide was shown for rat Leydig cells in primary culture. A polymerase chain reaction technique demonstrated the presence of functionally constituted vasopressin mRNA in rat and mouse testis, primary mouse Leydig cells and in rat and mouse Leydig tumour cell lines (MA10, R2C). Stimulation of cells with gonadotropins, however, had no effect either on peptide production or on levels of specific mRNA. Similarly, treatment of the MA10 cell line with a phorbol ester, or with rat atriopeptin, which activate other second messenger pathways, had no influence on vasopressin mRNA levels. The results are discussed in terms of an autocrine regulatory system which would provide the cell with information about its microenvironment.

Animals↗
PubMed1992

Is the relationship between angiogenesis and metastasis in breast cancer real?

The suggested link between angiogenesis in breast cancer and metastasis remains unsubstantiated. We tested this relationship in primary breast carcinomas from 37 patients with a median follow-up 9.5 years (Cohort 1) and 50 patients with a median follow-up of 1.5 years (Cohort 2). Angiogenesis was assessed by counting vessel density after immunohistochemical staining of vascular endothelium for factor VIII. Patients were grouped according to whether metastasis (defined as spread to axillary lymph nodes, distant sites or both) had occurred. The mean +/- SD scores in Cohort 1 when metastasis was absent and present, respectively, were 15.6 +/- 4.9 (n = 21) and 14.1 +/- 3.7 (n = 16). In Cohort 2 the scores were 15.4 +/- 5.8 (n = 26) and 14.5 +/- 4.9 (n = 24). There was no significant difference between these scores in either cohort. Multivariate analysis demonstrated lymph node involvement (P < 0.001) and tumour size (P < 0.001) but not angiogenesis score (P > 0.05) to predict distant metastasis. This evidence argues against any prognostic significance of angiogenesis in breast carcinoma.

Aged↗
PubMed1992

Mapping of a linear autoantigenic epitope within the human thyroid peroxidase using recombinant DNA techniques.

Autoantibodies directed against the thyroid peroxidase (TPO), the thyroid microsomal antigen, are widely used to diagnose human autoimmune thyroid disease. A cloned 3.088 kb cDNA coding for the entire mature human TPO was isolated from a cDNA library derived from a pathological thyroid gland of a Graves' disease patient and used further to generate a so-called TPO epitope cDNA library in order to map linear autoantigenic epitopes involving a recombinant molecular biology approach. The TPO epitope cDNA library consisting of randomly fragmented cDNA sequences inserted in the expression vector pGEX-2T was expressed in Escherichia coli and screened with characterized anti-TPO autoantisera from Hashimoto's disease patients. All the sera were positively tested with a purified thyroid microsomal antigen fraction (TMA/TPO). Only about 1% of examined autoantisera were able to recognize bacterial expressed recombinant TPO representing sequential antigenic determinants. A corresponding autoantigenic epitope with 61 amino acids in length was located at the C-terminus of human TPO.

Autoantibodies↗
PubMed1992

Season and manic relapse.

The episodes of mania occurring in a cohort of 86 patients with bipolar affective disorder were recorded over a 6-year period. Of the 54 patients who had at least 2 episodes, 8 (15%) met our criteria for a seasonal pattern of relapse. There was no particular season in which these relapses clustered. Overall, the time of year of the first recorded episode during the 6-year period did not predict the timing of subsequent episodes.

Bipolar Disorder↗
PubMed1991

Tyrosine hydroxylase polymorphisms and bipolar affective disorder.

A reported genetic association between bipolar affective disorder and DNA polymorphisms at the tyrosine hydroxylase gene is not confirmed by the present study. The combined allele frequencies in the patients, from studies published to date, are significantly different from the frequencies in the controls for the TY7/BglII polymorphism.

Alleles↗
PubMed1991

Monoclonal antibodies define linear and conformational epitopes of HIV-1 pol gene products.

Purified recombinant reverse transcriptase (RT) from human immunodeficiency virus type 1 (HIV-1) was used to raise 21 monoclonal antibodies with anti-RT specificities. The antibodies were characterized using Western blotting against native virus and recognized either the p66 or p66, p51 components of RT. Further immunoblotting using either cyanogen bromide fragmented RT or truncated mutants of RT along with cross-competition studies enabled the location of various immunogenic regions of RT to be identified. Three antibodies recognized a linear epitope in the N-terminal region (amino acids 128-176). Also, a neutralizing RT antibody recognized a conformational epitope in this region. Three monoclonals had epitopes mapped to linear sequences in the RNase H region at the C-terminus of the RT. Another neutralizing antibody, also requiring folding of the RT protein had its epitope more centrally located (231-353). Of the remaining 13 monoclonals, 7 were roughly located in the C-terminal region and required folding of the protein for epitope recognition and only three of the remaining six could be mapped to conformational epitopes in N-terminal and central regions of the RT. None of the antibodies tested recognized HIV-2 RT products p68 and p55 in Western blot.

Animals↗
PubMed1991

Tardive dyskinesia in bipolar affective disorder: a catchment area study.

The prevalence of tardive dyskinesia in a consecutive series of 69 patients with bipolar affective disorder admitted to a catchment area service was found to be 19%. Prevalence increased with age and was related to the age of onset of bipolar disorder and number of previous episodes. Patients with tardive dyskinesia were significantly slower on a simple test of cognitive function (Trails B).

Adult↗
PubMed1991

Tachykinin (substance-P) gene expression in Leydig cells of the human and mouse testis.

Specific substance-P immunoreactivity can be detected in the Leydig cells, particularly of human testes, and to a lesser degree in mouse Leydig cells, but not in the rat. Using a modified polymerase chain reaction (PCR) assay, preprotachykinin-A (substance-P) mRNA could be detected in extracts of human, mouse, and bovine testes, but not in rat or boar testes or in bovine thyroid or corpus luteum used as negative controls. This assay is able to discriminate among the alpha, beta, and gamma transcripts of the gene and shows that only the beta and gamma transcripts are present in the testes. Sequencing analysis of the PCR products from bovine hypothalamus, mouse brain, and human testis confirmed the structure of these transcripts, which encode both substance-P and neurokinin-A (substance-K) neuropeptide hormones. Using a variant of this assay it was possible to identify tachykinin transcripts in as few as 500 freshly prepared purified mouse Leydig cells. In parallel studies PCR analysis was also able to confirm the presence of mRNA for both substance-P and neurokinin-A receptors in human testes. Thus, the tachykinins substance-P and neurokinin-A must now be added to the list of potentially paracrine substances regulating intratesticular function.

Aged↗
PubMed1991

Decidual-type prolactin expression by the human myometrium.

The human myometrium, in addition to the decidualized endometrium of the late luteal phase and of pregnancy, has been proposed as a second source of uterine PRL, since immunoreactive PRL was found in supernatants from myometrial explant cultures. We demonstrate here that: 1) the human (h) PRL gene is expressed in the myometrium in vivo; 2) myometrial PRL is identical to pituitary hPRL; 3) the encoding transcript differs from pituitary hPRL messenger (m) RNA but is homologous to decidual and IM-9-P3 lymphoid hPRL mRNA; and 4) the expression of myometrial hPRL mRNA is inhibited by progestin. hPRL mRNA was detected in freshly isolated myometrium by Northern blot hybridization and was larger than the pituitary message. Sequence and primer extension analyses revealed that the transcript is identical to pituitary hPRL mRNA downstream of the pituitary cap site and carries an extension of the 5'-untranslated region homologous to that of decidual/IM-9-P3 lymphoid hPRL mRNA. This mRNA species results from alternative transcription initiation and comprises exon 1a of the hPRL gene, which is not transcribed in the pituitary. hPRL mRNA steady state levels and hPRL secretion increased dramatically when myometrial explants were maintained in long term culture. In addition to the 23,000 mol wt form, myometrial explants synthesized a glycosylated hPRL variant (G-hPRL) which was approximately 500 Daltons larger than pituitary G-hPRL but of similar size as lymphoid G-hPRL (26,500). Lactogenic activity of myometrial conditioned medium paralleled that of pituitary hPRL in the Nb2 lymphoma bioassay and was neutralized by the addition of monoclonal antibody to hPRL. hPRL secretion and hPRL mRNA abundance were not affected by estrogen but were markedly reduced by medroxy-progesterone acetate, which was maximally effective at a dose as low as 10(-10) M.

Adult↗
PubMed1990

Structure and ovarian expression of the oxytocin gene in sheep.

In sheep, the oxytocin gene is highly up-regulated in the ovarian corpus luteum as well as in the hypothalamus. This expression is already elevated on Day 2 of the oestrous cycle, representing 1% of all transcripts in this tissue, and it declines thereafter to low levels after Day 6 of the cycle. In order to study the mechanisms involved in luteal oxytocin gene expression, we have cloned and sequenced the oxytocin gene from the sheep. This gene is closely homologous to other known mammalian oxytocin genes, especially the bovine one, and comparison of the gene promoter regions highlights several blocks of putative control elements.

Amino Acid Sequence↗
PubMed1990

Seasonal affective disorder following brain injury.

Seasonal affective disorder has not previously been linked with neuroanatomical abnormalities despite its relationship to biological rhythms. A 45-year-old woman is described with an arteriovenous abnormality in the right frontotemporal region who developed recurrent winter depression and summer hypomania.

Brain Injuries↗
PubMed1989

Expression of the human relaxin gene in the corpus luteum of the menstrual cycle and in the prostate.

DNA-RNA hybridization has been used to assess the presence of relaxin gene transcripts in human luteal tissues of pregnancy and the menstrual cycle, as well as in the human testis and prostate. The results imply a substantial capacity for hormone biosynthesis in the mid to late luteal phase of the ovary in non-pregnant women. In men the prostate has been shown also to express relaxin gene transcripts, though levels are low. The testis appears negative. The results suggest that functions for relaxin must be sought also outside pregnancy.

Base Sequence↗