Melatonin responses to clonidine and yohimbine challenges.

Melatonin (MT) release from the pineal gland has been used as a marker for central noradrenergic function in major depression. Norepinephrine acts at both alpha and beta adrenergic receptors on the pinealocyte membrane to mediate nocturnal MT release, but in humans the contribution of each receptor class is unclear. In order to explore the effect of alpha 2 receptors on MT release, 10 female subjects were given oral challenges, in separate placebo-controlled trials, of either 10.8 mg of yohimbine, an alpha 2 adrenergic antagonist, or clonidine, a partial alpha 2 adrenergic agonist, at doses of either 200 micrograms or 300 micrograms. Post-challenge serum melatonin was measured from 18:00 h to 22:00 h in both studies as was urinary 6-sulphatoxy-melatonin (aMT6s), the major metabolite of MT (from 18:00 h to 22:00 h, and from 22:00 h to 10:00 h). Growth hormone (GH) was also assayed following the clonidine challenge, and blood pressure, pulse rate, and side effects were monitored after both challenges. Neither yohimbine nor clonidine significantly altered nocturnal serum MT levels compared to placebo. However, there was a significant increase in urinary aMT6s between 18:00 h and 22:00 h following yohimbine ingestion. Yohimbine ingestion produced significant rises in pulse rate and the urge to urinate compared to placebo. Both doses of clonidine resulted in a significant reduction in pulse rate, systolic and diastolic blood pressure, and significant increases in drowsiness and other measures of sedation following ingestion. Only clonidine 300 micrograms produced a significant elevation in GH release. This study highlights the limitations of oral neuroendocrine challenge studies.     

Validation of computerized three-dimensional reconstruction of intravascular ultrasound: measurements of absolute luminal diameter and cross-sectional area in ex vivo human coronary arteries

Computer based 3-dimensional reconstruction transforms 2-dimensional intravascular ultrasound images into a longitudinal format facilitating analysis of luminal narrowing. To validate the accuracy of current software in measuring coronary artery diameter and cross-sectional area, in arteries with atherosclerosis, we performed 3-dimensional reconstruction in 10 human pathologic coronary arterial segments of 10-25mm length. Images were obtained using a 4.8 French catheter with pullback speed of 1mm/sec acquired at 3 frames/sec onto VHS tape. The data were digitized and intraluminal 3-dimensional reconstruction performed using a voxel-based program. Pathologic sections were obtained every 3mm, and dimensions were measured with a resolution of 0.01 mm. Maximum, minimum, and 3 other representative diameters were recorded by an observer blinded to the ultrasound diameters. Average histo-pathologic diameters were reported, and specimen cross-sectional area was then calculated. Results: In 53 sections, pathological diameters ranged from 1.4-4.5mm (mean 2.7 +/- 0.68mm) while 3-dimensional reconstructed diameters were 1.9 to 3.8mm (mean 2.6 +/- 0.54mm). Pathologic and ultrasound derived 3-dimensional reconstruction diameters had an excellent correlation (r=0.86, SEE=+/-0.36). Pathology and 3-dimensional reconstruction cross-sectional area also correlated closely (r=0.88, SEE=+/-1.50). Diameters less than 2.0mm were systematically overestimated and diameters greater than 3.5mm underestimated by 3-dimensional reconstruction. Most 3 dimensional reconstruction values were within +/- 10% of pathology, but diverged at each diameter extreme, approaching +/- 20%. Thus, computerized 3-dimensional reconstruction of ultrasound images shows excellent quantification of luminal size in the 2.0-3.5mm range, suggesting important investigative and clinical applications.     

Polymorphisms of the equine major histocompatibility complex class II DRA locus

The full extent of the polymorphism of ELA-DRA in Equidae is not yet known. Given the apparent differences in DRA polymorphisms between Equidae and other species, the aims of this study were to more fully characterize ELA-DRA, determine the extent of gene polymorphism and establish the allele-frequency distribution. An allele reference panel for the second exon of ELA-DRA was established by sequence-based typing of 69 equine DNA samples consisting of various breeds of domestic horse (Equus caballus), together with donkeys (Equus asinus), Grant's zebras (Equus boehmi) and one onager (Equus hemionus). Five of the six previously reported alleles detected using single-strand conformation polymorphism were found: ELA-DRA*0101, ELA-DRA*0201, ELA-DRA*0301, ELA-DRA*0501 (Albright-Fraser DG et al. Polymorphism of DRA among equids. Immunogenetics 1996: 43: 315-7) and ELA-DRA*0601 (GenBank accession number AF5419361). In addition to the previously reported alleles, five novel ELA-DRA alleles were detected within the ELA-DRA allele reference panel. One of these was identified in E. caballus (ELA-DRA*JBH11), one in E. boehmi and E. hemionus (ELA-DRA*JBZ185) and three in E. asinus (ELA-DRA*JBD3, ELA-DRA*JBD17 and ELA-DRA*JBH45). A total of 565 equine DNA samples were screened using reference-strand-mediated conformation analysis, a double-stranded conformation-based mutation detection system that can be used to type existing ELA-DRA alleles and identify new variants. Based on our findings, at least 11 ELA-DRA alleles are now known to exist, and this level of polymorphism at the DRA locus appears to be unique to the genus Equus. Both the previously reported alleles and the new alleles displayed a species-specific distribution.     

An assessment of the delayed hypersensitivity reaction to methylated bovine serum albumin in the mouse and its use in the evaluation of drug effects on cell mediated immune reactions

A delayed hypersensitivity (DH) reaction is induced by a sensitizing intradermal injection of methylated bovine serum albumin (MBSA) into the abdomen of mice and a subsequent challenge injection of MBSA into the hind paw. Paw volume increase is measured by mercury plethysmography. The conditions for sensitization have been investigated. Sensitization with a 0.25% MBSA emulsion resulted in a small but significant swelling of the paw following the challenge injection. The magnitude of the footpad response to the challenge injection was increased if the antigen administered in the sensitizing injection was emulsified with Freund's incomplete adjuvant. Incorporation ofMycobacterium butyricum in the emulsion greatly increased the footpad response if added at doses of 0.05 and 1 mg per animal. A higher dose (4 mg), however, resulted in a lower response. The time course of development of the delayed hypersensitivity reaction has been studied. An 8-day interval between sensitization and challenge resulted in a greater delayed hypersensitivity response than a shorter (3-day) or longer (15-, 21-, 28-day) interval. Cyclophosphamide (250 mg kg^–1) administered 3 days prior to the sensitizing injection of MBSA produced a modest enhancement of the DH reaction.On the basis of these studies a protocol for conducting the DH reaction to MBSA was established and the activity of drugs on processes underlying the sensitization phase of the reaction or processes underlying the elicitation phase of the reaction have been examined. Steroid and immunosuppressant drugs were found to inhibit the DH footpad response when dosed during the sensitization whereas several specific-anti-rheumatic and immunoactive compounds were without effect. Indomethacin and sudoxicam inhibited the DH reaction if dosed during the elicitation of the reaction but other non-steroidal anti-inflammatories tested did not significantly reduce the response. The clinically used anti-rheumatic drugsd-penicillamine and levamisole did not inhibit the elicitation phase of the DH response but niridazole at 100 mg kg^–1 did reduce the inflammatory response.This paper has been presented in part as a poster presentation to the British Pharmacological Society, 4–6 January 1978.     

Sequence variation in the 3'-untranslated region of the dopamine transporter gene and attention-deficit hyperactivity disorder (ADHD).

The dopamine transporter gene (DAT1) has been reported to be associated with attention-deficit hyperactivity disorder (ADHD) in a number of studies [Cook et al. (1995): Am J Human Genet 56(4):9993-998; Gill et al. (1997): Mol Psychiatry 2(4):311-313; Waldman et al. (1998): Am J Human Genet 63(6):1767-1776; Barr et al. (2001): Biol Psychiatry 49(4):333-339; Curran et al. (2001): Mol Psychiatry 6(4):425-428; Chen et al. (2003): Mol Psychiatry 8(4):393-396]. Specifically, the 10-repeat allele of the 40-bp variable number of tandem repeats (VNTR) polymorphism located in the 3' untranslated region (UTR) of the gene has been found to be associated with ADHD. There is evidence from in vitro studies indicating that variability in the repeat number, and sequence variation in the 3'-UTR of the DAT1 gene may influence the level of the dopamine transporter protein [Fuke et al. (2001): Pharmacogenomics J 1(2):152-156; Miller and Madras (2002): Mol Psychiatry 7(1):44-55]. In this study, we investigated whether DNA variation in the DAT1 3'UTR contributed to ADHD by genotyping DNA variants around the VNTR region in a sample of 178 ADHD families. These included a MspI polymorphism (rs27072), a DraI DNA change (T/C) reported to influence DAT1 expression levels, and a BstUI polymorphism (rs3863145) in addition to the VNTR. We also screened the VNTR region by direct resequencing to determine if there was sequence variation within the repeat units that could account for the association. Our results indicate that DAT1 is associated with ADHD in our sample but not with alleles of the VNTR polymorphism. We did not find any variation in the sequence for either the 10- or 9-repeat alleles in the probands screened nor did we observe the reported DraI (T/C) variation. Our results therefore refute the possibility of the reported DraI variation or alleles of the VNTR as the functional variants contributing to the disorder.     

No association between the Pro197Leu polymorphism in the glutathione peroxidase (GPX1) gene and schizophrenia

OBJECTIVE: Oxidative stress such as free radical-mediated neuronal dysfunction may be involved in the pathophysiology of schizophrenia. The human glutathione peroxidase (GPX1) is a selenium-dependent enzyme, which plays an important role in the detoxification of free radicals. We therefore hypothesized that the GPX1 gene, which is located on chromosome 3p21.3, may be involved in the pathophysiology of schizophrenia. The aim of this study is to examine whether a potentially functional polymorphism, a proline (Pro) to leucine (Leu) substitution at codon 197 (Pro197Leu) of the human GPX1 gene, is associated with susceptibility to schizophrenia. METHODS: We genotyped the Pro197Leu polymorphism in a total of 113 nuclear families that had a proband with schizophrenia. Genetic association was tested using the transmission disequilibrium test (TDT), the sib transmission disequilibrium test (STDT), and the family-based association test (FBAT). RESULTS: The minor allele (Leu) frequency was calculated to be 0.282. We could not find significant transmission disequilibrium of the alleles for the Pro197Leu polymorphism in the GPX1 gene in association with the presence of schizophrenia in our family sample (TDT, chi2=0.03, degrees of freedom=1, P=0.86; combined TDT-STDT, Z'=-0.052, P=0.47; FBAT, Z=0.000, P=1.000). CONCLUSION: The results of this study suggest that the GPX1 polymorphism is unlikely to be associated with susceptibility to schizophrenia.     

Association study of dopamine D3 receptor gene and schizophrenia

Several groups have reported an association between schizophrenia and the MscI polymorphism in the first exon of the dopamine D3 receptor gene (DRD3). We studied this polymorphism using a North American sample (117 patients plus 188 controls) and an Italian sample (97 patients plus 64 controls). In the first part of the study, we compared allele frequencies of schizophrenia patients and unmatched controls and observed a significant difference in the total sample (P = 0.01). The second part of the study involved a case control approach in which each schizophrenia patient was matched to a control of the same sex, and of similar age and ethnic background. The DRD3 allele frequencies of patients and controls revealed no significant difference between the two groups in the Italian (N = 53) or the North American (N = 54) matched populations; however, when these two matched samples were combined, a significant difference was observed (P = 0.026). Our results suggest that the MscI polymorphism may be associated with schizophrenia in the populations studied. © 1995 Wiley-Liss, Inc.