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Biomedical subjects

N Hunt

Publications and source records attributed to N Hunt.

At least 19 recordsLinked to original sources
PubMed1998

Enhanced high density oligonucleotide array-based sequence analysis using modified nucleoside triphosphates.

Pairs of high density oligonucleotide arrays (DNA chips) consisting of >96 000 oligonucleotides were designed to screen the entire 5.53 kb coding region of the hereditary breast and ovarian cancer BRCA1 gene for all possible sequence changes in the homozygous and heterozygous states. Single-stranded RNA targets were generated by PCR amplification of individual BRCA1 exons using primers containing T3 and T7RNA polymerase promoter tails followed by in vitro transcription and partial fragmentation reactions. Fluorescent hybridization signals from targets containing the four natural bases to >5592 different fully complementary 25mer oligonucleotide probes on the chip varied over two orders of magnitude. To examine the thermodynamic contribution of rU.dA and rA.dT target.probe base pairs to this variability, modified uridine [5-methyluridine and 5-(1-propynyl)-uridine)] and modified adenosine (2,6-diaminopurine riboside) 5'-triphosphates were incorporated into BRCA1 targets. Hybridization specificity was assessed based upon hybridization signals from >33 200 probes containing centrally localized single base pair mismatches relative to target sequence. Targets containing 5-methyluridine displayed promising localized enhancements in hybridization signal, especially in pyrimidine-rich target tracts, while maintaining single nucleotide mismatch hybridization specificities comparable with those of unmodified targets.

Base Pair Mismatch↗
PubMed1998

Function of the nasal muscles in normal subjects assessed by dynamic MRI and EMG: its relevance to rhinoplasty surgery.

The external nose is generally considered to have a relatively static shape. Movement of the nose and the potential for change of external shape and the internal airway have been assessed by (1) cadaver dissection and (2) dynamic studies during a standardized series of facial expressions in 13 subjects; video recording of the movement of skin markers, electromyography, and moving magnetic resonance images. A standard description of muscle anatomy is presented. The dynamic investigations indicated the following. Video analysis showed the components of muscle action: dilatation, constriction of the nostril, depression of the tip, vertical contraction in the bridge, and elevation of the alar, in different expressions. Electromyography confirmed muscle actions during expression and phonation. Magnetic resonance imaging demonstrated large changes in the external shape of the nose and nasal aperture due to muscle actions. Muscle function should be given greater consideration in aesthetic and cleft rhinoplasty.

Electromyography↗
PubMed1998

Strategies for mutational analysis of the large multiexon ATM gene using high-density oligonucleotide arrays.

Mutational analysis of large genes with complex genomic structures plays an important role in medical genetics. Technical limitations associated with current mutation screening protocols have placed increased emphasis on the development of new technologies to simplify these procedures. High-density arrays of >90,000-oligonucleotide probes, 25 nucleotides in length, were designed to screen for all possible heterozygous germ-line mutations in the 9.17-kb coding region of the ATM gene. A strategy for rapidly developing multiexon PCR amplification protocols in DNA chip-based hybridization analysis was devised and implemented in preparing target for the 62 ATM coding exons. Improved algorithms for interpreting data from two-color experiments, where reference and test samples are cohybridized to the arrays, were developed. In a blinded study, 17 of 18 distinct heterozygous and 8 of 8 distinct homozygous sequence variants in the assayed region were detected accurately along with five false-positive calls while scanning >200 kb in 22 genomic DNA samples. Of eight heterozygous sequence changes found in more than one sample, six were detected in all cases. Five previously unreported sequence changes, not found by other mutational scanning methodologies on these same samples, were detected that led to either amino acid changes or premature truncation of the ATM protein. DNA chip-based assays should play a valuable role in high throughput sequence analysis of complex genes.

Ataxia Telangiectasia Mutated Proteins↗
PubMed1998

Microvascular and cellular responses in the optic nerve of rats with acute experimental allergic encephalomyelitis (EAE).

The optic nerve of rats with EAE was examined at various times to determine the integrity of the blood-brain barrier (BBB) and to assess monocyte-macrophage, T cell, and microglial responses. In naive control animals, leakage of horseradish peroxidase (HRP) and the presence of cells expressing major histocompatibility complex (MHC) class II antigen were evident in the meninges of the retrobulbar optic nerve. In rats with EAE, microglia in the region of the lamina cribrosa and in the regions adjacent to the meninges became activated from day 7 to 8 postinduction (pi). HRP leakage was also evident in the region of the lamina cribrosa from day 7 to 8 pi. On day 8 pi, infiltration of inflammatory cells and Monastral blue leakage were apparent in the myelinated region of the optic nerve. The intensity of these cellular and vascular changes peaked at day 12 pi, when signs of clinical disease became manifest. Monocytes-macrophages expressing MHC class II and the ED1 antigen, together with lymphocytes expressing the alphabetaT cell receptor, constituted the major proportion of cells associated with inflammatory lesions. Thus: (i) the inherent weakness of the BBB as well as the presence of both antigen (myelin) and MHC class II+ cells in the retrobulbar optic nerve are likely susceptibility factors for the frequent involvement of this region in EAE and multiple sclerosis; and (ii) activation of microglia occurs early in the pathogenesis of experimental optic neuritis.

Animals↗
PubMed1998

Microvascular and cellular responses in the retina of rats with acute experimental allergic encephalomyelitis (EAE).

The microvascular and cellular responses in the retina during acute EAE were characterized using whole-mount preparations. The earliest detectable event was the accumulation of monocytes and T cells within veins on day 7 postinduction (pi). Mild breakdown of the blood-retinal barrier (BRB), activation of microglia and infiltration of monocytes and T cells into the retinal parenchyma were first evident on days 7 to 8 pi. Monocyte adhesion to the vessel wall and breakdown of the BRB were colocalized in the same vessel segments and occurred predominantly in veins. The marked difference in response observed in the retina versus the myelinated region of the optic nerve suggests that two types of inflammatory cascades are initiated. A mild response, characterised by very low numbers of T cells and monocytes and an absence of expression of MHC class II by resident microglia, is initiated when only small amounts of the encephalitogenic antigen are present in the perivascular space or associated with perivascular antigen-presenting cells. A full blown inflammatory reaction, as observed in the optic nerve, is initiated in the presence of substantial amounts of encephalitogenic antigen. This severe response is characterised by the infiltration of large numbers of CD4+, CD8+ T cells and ED1+ monocytes, and by abundant MHC class II expression by resident microglia as well as other cell types. Thus, MHC class II expression by resident microglia may be a possible effective amplifier mechanism if the encephalitogenic antigen is encountered in the tissue parenchyma.

Animals↗
PubMed1998

cis-Active elements of Friend spleen focus-forming virus: from disease induction to disease prevention.

The polycythemic strain of the Friend spleen focus-forming virus (SFFVp) is a replication-defective, acutely transforming retrovirus inducing a bistage erythroleukemia in susceptible mice. The first stage of the disease is an acute polyclonal erythroblastosis induced by the proliferation-promoting effect of gp55. gp55 is expressed from a spliced subgenomic message of SFFVp and stimulates the cellular receptor for erythropoietin. Using a selectable SFFVp that otherwise mimics the specificity of the disease, we demonstrate that the kinetics of the polyclonal expansion depends on the transcriptional strength of the retroviral cis-active elements. By exchanging gp55 for apathogenic genes, we show that SFFVp enhancer and splice signals can be successfully utilized for the development of retroviral vectors mediating very efficient transgene expression in hematopoietic cells. Apathogenic selectable SFFVp-based vectors carrying distinct enhancer alterations are a valuable tool to analyze transcriptional control of leukemia viruses in the absence of oncogenic proteins. Moreover they might have therapeutic potential.

Animals↗
PubMed1998

Relaxin-like factor expression as a marker of differentiation in the mouse testis and ovary.

Expression of the relaxin-like factor (RLF) was studied at the messenger RNA (mRNA) and protein levels in the testes and ovaries of the mouse, as well as through testicular development and differentiation in the mouse testis. In situ hybridization or RT-PCR, and immunohistochemistry using a polyclonal antibody raised against a recombinant protein, provided mutually confirmatory results for a high expression of RLF in the Leydig cells of the adult testis and at a much lower level of expression in the luteal cells of the ovary through the cycle, pregnancy, and in lactation. Analysis of protein and mRNA expression, through postnatal testicular development, indicated moderate RLF expression also in the fetal population of Leydig cells, even in the hpg mutant mouse, lacking an active pituitary-gonadal axis. Prepubertal Leydig cells, however, exhibit only very low-level RLF gene expression, this phenotype persisting in the adult hpg mouse. In summary, fetal Leydig cells express RLF in an LH/human CG-independent fashion, whereas LH/human CG is essential to induce RLF expression in the adult-type Leydig cell. In cultured adult Leydig cells or in the mouse tumor MA-10 cell line, RLF mRNA is expressed in a constitutive fashion. RLF thus seems to be a useful marker of Leydig cell differentiation status.

Animals↗
PubMed1998

Dependence on computer games by adolescents.

As computer game playing is a popular activity among adolescents, a questionnaire study was undertaken with 387 adolescents (12-16 years of age) to establish their "dependence" using a scale adapted from the DSM-III-R criteria for pathological gambling. Analysis indicated that one in five adolescents were currently "dependent" upon computer games. Boys played significantly more regularly than girls and were more likely to be classified as "dependent." The earlier children began playing computer games it appeared the more likely they were to be playing at "dependent" levels. These and other results are discussed in relation to research on other gaming dependencies.

Adolescent↗
PubMed1998

How effective is lithium in the prevention of relapse in bipolar disorder? A prospective naturalistic follow-up study.

OBJECTIVE: The effectiveness of lithium in preventing recurrences of bipolar disorder was examined prospectively for 2 years in two representative samples of bipolar I patients being treated in a comprehensive program following recommended guidelines. METHOD: One hundred and twenty patients were recruited from consecutive admissions to two catchment area psychiatric services (one in the United Kingdom, the other in New Zealand). They were seen at 3-monthly intervals by a member of the research team. Treatment was adjusted according to clinical needs. RESULTS: Overall, two-thirds of the patients had a recurrence. Of the 57 on lithium as sole treatment, 39 (68%) had a further episode; 17 after stopping lithium. The 42 on other mood stabilisers and/or an antipsychotic, with or without lithium, did no better. By contrast, only eight (38%) of the 21 who were taking no prophylactic medication had a recurrence. CONCLUSIONS: Lithium is much less effective in clinical practice than would be expected from clinical trial results. A major reason for this is poor compliance. Alternative treatment strategies are needed to improve the outcome for bipolar disorder patients.

Adolescent↗
PubMed1997

Relaxin-like factor: a highly specific and constitutive new marker for Leydig cells in the human testis.

The complete protein-coding region of the human relaxin-like factor (RLF; formerly Ley-I-L) was cloned by reverse transcription-polymerase chain reaction from human testis and subcloned into a bacterial expression plasmid for the production of recombinant human RLF in Escherichia coli. Polyclonal antibodies were raised against the recombinant RLF, as well as against a peptide epitope from the B-domain of the RLF polypeptide. Antibodies were used for immunohistochemistry of Bouin-fixed, paraffin-embedded samples of human testis tissues. Specific immunoreactivity was located exclusively in the Leydig cells with a consistent high intensity of staining, showing similar spatial distribution to other Leydig cell markers, such as the luteinizing hormone (LH) receptor and 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), and to the pattern of RLF mRNA shown by in-situ transcript hybridization. In biopsy samples from patients with severe disturbances of spermatogenesis, RLF staining intensity was consistently high in all cases, unlike staining for 3 beta-HSD which varied considerably between patients. Immunostaining for RLF would thus appear to offer an interesting new marker for Leydig cells in human testis samples.

3-Hydroxysteroid Dehydrogenases↗
PubMed1997

Neurophysiologic actions and neurological consequences of veratridine on the rat sciatic nerve.

BACKGROUND: The quest for a drug that would provide analgesia with minimal motor deficiency, through the selective inhibition of impulses in small-diameter fibers, was brightened by a previous report of veratridine's C-fiber-selective actions on the isolated rabbit vagus nerve. The goal of the present research was to demonstrate the same actions on rat sciatic nerve in vitro and to observe the functionally differential blockade in the rat in vivo. METHODS: Sciatic nerves were removed from rats, mounted in a recording chamber, wherein a 1-cm length of the ensheathed nerve was superfused with the plant alkaloid veratridine (2 microM) in bicarbonate-buffered Liley's solution, and the compound action potential (CAP) was stimulated supramaximally to give A- and C-fiber elevations. Onset, steady-state, and recovery from veratridine effects were assayed for a range of stimulus frequencies. Open-field behavior and quantitative neurological assessments of proprioception, motor function, and nociception were tested in 15 trained rats after injection near the sciatic nerve of 0.1 ml veratridine at 0.5, 0.7, and 1.0 mM each plus epinephrine (1:200,000). RESULTS: Veratridine inhibited the C-fiber component of the CAP in a frequency-dependent manner. At 0.1 Hz the CAP was 65% of the control amplitude, 50% at 0.5 Hz, and 40% at 5 Hz. A-fiber elevations were unattenuated at stimulus frequencies as high as 50 Hz. Steady-state inhibition was reached 5 min after drug administration, and recovery from the effects was 30% complete by 15 min of drug washout. Proprioception, measured as a "hopping" or "placing" reaction, was inhibited dose dependently by maximum degree and for durations of, respectively, 0.5 mM, 61%, 180 min; 0.7 mM, 100%, 360 min; and 1 mM, 100%, 420 min. Extensor postural thrust, as a measure of motor function, was inhibited by and for 0.5 mM, 77%, 240 min; 0.7 mM, 99%, 390 min; and 1 mM, 100%, 420 min. Analgesia, as a prolonged withdrawal latency to a noxious thermal stimulus, had the following profile: 0.5 mM, 10%, 30 min; 0.7 mM, 52%, 150 min; and 1 mM, 66%, 150 min. CONCLUSIONS: Despite the fact that veratridine gave a C-fiber preferential blockade in the isolated sciatic nerve, heightened analgesia over motor block was not achieved in vivo. Indeed, just the opposite occurred. If preferential C-fiber blockade also occurs in vivo, then its traditionally expected correlation with analgesia must be re-examined.

Action Potentials↗
PubMed1997

Analysis of human thyrotropin receptor gene expression and immunoreactivity in human orbital tissue.

The human thyrotropin receptor (hTSHR) represents an autoantigen that plays a central role in the hyperthyroidism of Graves' disease (GD). hTSHR transcripts have recently been detected by reverse transcription (RT)-PCR in various extrathyroidal tissues, suggesting that the hTSHR may be more widely distributed than previously thought, and that it may serve as a common antigen in the thyroidal and extrathyroidal manifestations of GD. Using techniques other RT-PCR, we examined whether RNA encoding hTSHR and hTSHR-specific immunoreactivity can be detected and localized in cultured orbital fibroblasts (OF), orbital connective tissue, extraocular muscle and various extraorbital tissues derived from both patients with Graves' ophthalmopathy (GO) and normal individuals. Using in situ hybridization with a digoxigenin-labeled antisense oligonucleotide probe specific for the extracellular domain of hTSHR, specific perinuclear and cytoplasmic hTSHR gene expression was detected in OF of patients with GO and, to a lesser degree, normal individuals. Using a highly sensitive immunostaining technique and a panel of monoclonal and polyclonal antibodies directed against different epitopes of recombinant hTSHR, distinct cytoplasmic hTSHR-like immunoreactivity was detected in methanol-fixed OF and orbital connective tissue, which was absent in abdominal fibroblasts, or when using isotype-matched non-immune immunoglobulins. Mouse monoclonal and pig polyclonal hTSHR antibodies detected cytoplasmic hTSHR-like immunoreactivity in perimysial fibroblasts within extraocular muscle, but not in extraocular muscle fibers. Immunocytochemical staining with rabbit polyclonal hTSHR antibody revealed, in addition, distinct cell surface-associated immunoreactivity in paraformaldehyde-fixed OF, but not in abdominal fibroblasts. Taken together, our results suggest that RNA encoding hTSHR is present and actively processed to immunoreactive hTSHR-like protein in OF residing within orbital connective tissue and extraocular muscle. These data support the concept that OF expressing intact or variant hTSHR may act as extrathyroidal targets for sensitized T-cells and immunoglobulins in GD.

Animals↗
PubMed1996

A novel endozepine-like peptide (ELP) is exclusively expressed in male germ cells.

A cDNA clone encoding a novel endozepine-like peptide (ELP) was isolated from mouse testes, sequenced, and its mRNA expression characterized by northern and in situ hybridization. ELP mRNA was found exclusively in the late spermatid stages of spermatogenesis in the testes of sexually mature mice and in no other tissue or cell type examined. It was also expressed in rat, bovine, porcine and sheep testes. Mouse ELP-encoding cDNA was used to construct expression vectors for the production of ELP in bacteria, and the purified bacterial protein used to raise polyclonal antibodies in rats. These antibodies identified the predicted endogenous ELP in extracts of mouse testis and epididymis and in no other tissue. Immunohistochemistry confirmed that the ELP antigen was present only in late spermatids and spermatozoa, particularly within the cytoplasmic droplet which is retained by the mature spermatozoa during their transit into the epididymis. We conclude that ELP is an intracytoplasmic peptide exclusively expressed in post-meiotic spermatozoa and which may be involved in the energy metabolism of the mature sperm.

Amino Acid Sequence↗
PubMed1996

Epitope mapping of a recombinant human TSH receptor extracellular domain: identification of a predominant epitope using animal sera.

The extracellular domain of the TSH receptor (TSHR-561, amino acids #78-389) was expressed as a hexa-histidine fusion protein in bacteria. The recombinant protein was purified to homogeneity and used to immunize porcine and ovine species. High titre antibodies were obtained from both species that recognized the recombinant protein in Western blot analysis but failed to interfere with the TSH radio receptor assay. An epitope library was constructed and screened with affinity purified ovine and porcine antisera and detected a number of positive clones. Sequence analysis revealed that all of the epitopes contained sequences derived from the carboxyl terminus of the recombinant immunogen. One clone defined an epitope covering 16 amino acids from the carboxyl terminus and was the common epitope found in all of the other clones. Western blot screening of a large panel of Graves' sera with recombinant TSH receptor protein identified one patient sera that also recognized linear epitopes in the TSHR-561 protein. Experimentation demonstrated that the linear epitope recognized by this human sera was identical to the sequence recognised by the animal antisera. This sequence is unique to the TSH receptor and will be useful in further studies to analyze the TSH receptor protein.

Amino Acid Sequence↗
PubMed1996

The antibodies causing thyroid stimulating hormone-binding inhibition (TSH-BI) are not responsible for the specific inhibition of gonadal steroidogenesis by Graves' sera.

Graves' disease is attributed to the presence of autoantibodies with agonist activity which interact with the TSH receptor causing thyroid hyperstimulation and hyperthyroidism. The degree of TSH-binding inhibition (TSH-BI) caused by a Graves' serum in a TSH radioligand receptor assay is considered to be an index of the prevalence of anti-TSH receptor autoantibodies in that serum. We have previously shown that the specific inhibition by Graves' serum of hCG-stimulated steroidogenesis by Leydig cells was at a site distal to receptor binding and second messenger activation. In this report, we have investigated whether the effect of Graves' serum upon Leydig cells is a property of the constitutive antibodies. Immunoglobulin-enriched fractions were obtained from Graves' and normal sera using three increasingly rigorous procedures; ammonium sulphate precipitation, caprylic acid treatment and Protein A or G-affinity purification. The TSH-BI was determined for untreated and extracted sera in two radioreceptor assays developed for use with serum, one using human thyroid membranes and the other using HeLa cells transfected with the human TSH receptor, and the results were compared with effects in the Leydig cell steroidogenesis bioassay. The specific inhibition of hCG-stimulated Leydig cell steroidogenesis by Graves' sera was not retained in the antibody fraction causing TSH-BI. Thus, the inhibitory factor appears not to be an antibody and we are now attempting to purify and identify the responsible factor from Graves' serum.

Ammonium Sulfate↗
PubMed1996

Epitope mapping of the human TSH receptor; structure function studies.

With the aid of recombinant DNA technology (PCR/site directed mutagenesis, sequencing) the full length coding region of the human TSH receptor was manipulated to place a specific epitope peptide tag (FLAG epitope sequence) at the carboxyl end of the protein. The resulting construct was cloned into a eukaryotic expression vector and stably transfected into HeLa cells. The expression/translation of the tagged TSH receptor molecule was monitored by immune-precipitation and western blotting of protein lysates, and was found to be expressed at considerable levels using the commercially available antibodies directed towards the FLAG epitope. This analysis revealed two discrete specific bands 90-120 KDa representing, presumably, differently glycosylated forms of the receptor. TSH radio receptor assays demonstrated that the FLAG tagged TSH receptor bound TSH comparable with the wild type receptor. Furthermore TSH stimulated cAMP response in these transfected cells were comparable to the wild type receptor, thus demonstrating that the tagged receptor was functionally identical to the transfected wild type receptor. These cell lines will be of great value when analysing TSH/receptor or receptor/autoantibody interactions considering the availability of well characterized experimental anti-TSH receptor sera.

Blotting, Western↗