Selective pituitary resistance to thyroid hormone produced by expression of a mutant thyroid hormone receptor beta gene in the pituitary gland of transgenic mice

Phosphine (PH3), from hydrolysis of metal phosphides, is an important insecticide (aluminum phosphide) and rodenticide (zinc phosphide) and is considered genotoxic and cytotoxic in mammals. This study tests the hypothesis that PH3-induced genotoxicity and cytotoxicity are associated with oxidative stress by examining liver (Hepa 1c1c7) cells for possible relationships among cell death, increases in reactive oxygen species (ROS) and lipid peroxidation, and elevated 8-hydroxyguanine (8-OH-Gua) in DNA. PH3 was generated from 0.5 mM magnesium phosphide (Mg3P2) to give 1 mM PH3 as the nominal and maximal concentration. This level causes 31% cell death at 6 h, measured by lactate dehydrogenase leakage, with appropriate dependence on concentration and time. The intracellular ROS level is elevated within 0.5 h following exposure to PH3, peaking at 235% of the control by about 1 h. Lipid peroxidation (measured as malondialdehyde plus 4-hydroxyalkenals) is increased up to 504% by PH3 at 6 h in a time-dependent manner. The level of 8-OH-Gua in DNA, a biomarker of mutagenic oxidative DNA damage analyzed by GC/MS, increases to 259% at 6 h after PH3 treatment. Antioxidants significantly attenuate the PH3-induced ROS formation, lipid peroxidation, 8-OH-Gua formation in DNA, and cell death, with the general order for effectiveness of GSH (5 mM) and D-mannitol (10 mM) (hydroxyl radical scavengers), then Tempol (2.5 mM) and sodium azide (3 mM) (superoxide anion and singlet oxygen scavengers, respectively). These studies support the hypothesis that PH3-induced mutagenic and cytotoxic effects are due to increased ROS levels, probably hydroxyl radicals, initiating oxidative damage.     

Mutation analysis of the gonadotropin-releasing hormone receptor gene in idiopathic hypogonadotropic hypogonadism

The chemical signals of mammalian skin that stimulate the secretion of acetabular gland contents of Schistosoma mansoni cercariae were determined by exposing cercariae to fractions of human and pig skin surface obtained by thin-layer chromatography. Postacetabular gland secretion was stimulated by hydrophilic skin extracts but was often combined with a secretion of preacetabular glands. Secretion of preacetabular glands, which contain enzymes for skin lysis, could be selectively stimulated with skin surface lipids. Two different mechanisms of lipid-stimulated preacetabular gland release could be distinguished. First, secretion in combination with penetration behavior and probably tegument transformation was stimulated by the fraction of free fatty acids. Second, secretion independent of penetration behavior and tegument transformation was exclusively stimulated by glucosylceramides and phospholipids, probably phosphatidylcholines. The secretion mechanisms seem to allow a continuous lysis of epidermal macromolecules during the skin passage of the cercariae. Free fatty acids occur in the uppermost skin layers and may stimulate the combination of the first response; phospholipids and glucosylceramides are restricted to deeper epidermal layers and may stimulate the enzyme secretion there. An active preacetabular gland release was also stimulated by toxic chemicals, which could suggest an emergency penetration program for impaired cercariae.     

A functionally significant allelic polymorphism in a T cell receptor V beta gene segment

The effect of an allelic polymorphism in the BV1S1 gene segment on recognition of major histocompatibility complex (MHC)-peptide complexes by a specific T cell receptor (TCR) was studied using RBL 2H3 cells transfected with TCR-CD3 zeta chimeric receptors. An HLA-A2-restricted human immunodeficiency virus (HIV) pol-specific cytotoxic T lymphocyte (CTL) clone utilizing the BV1S1A2 gene in combination with AV2S1A2 was identified and the extracellular domains of the TCR were fused to CD3 zeta. In degranulation assays RBL 2H3 transfectants expressing this receptor maintained the specificity of the parental CTL clone. The allelic variant BV1S1A1N1 containing a glutamine for histidine substitution at position 48 in the loop of the second complementarity-determining region was generated by site-directed mutagenesis. Transfection of this molecule as a CD3 zeta chimera together with the original AV2S1A2 CD3 zeta molecule resulted in cell surface expression of both chains but a loss of recognition of HLA-A2 HIV pol peptide-pulsed targets. The effect of this polymorphism on MHC-peptide recognition supports current models of TCR MHC-peptide interaction and provides evidence for a functional role for polymorphism in the TCRV genes.     

Two novel mutations in the thyrotropin (TSH) receptor gene in a child with resistance to TSH

The TSH receptor is a G protein-coupled receptor that mediates the effects of TSH in thyroid development, growth, and synthetic function. We report here that a child with features of TSH resistance, including markedly increased serum TSH concentrations and low normal thyroid hormone levels, is a compound heterozygote for two novel mutations in the TSH receptor gene. One allele has a G to A transition corresponding to an arginine to glutamine change at codon 109 (R109Q) in the extracellular domain of the receptor. The other allele has a G to A transition corresponding to a premature termination codon at tryptophan 546 (W546X) in the fourth transmembrane segment. Each parent is heterozygous for one mutation, and both parents have normal thyroid function. Cells transiently transfected with the R109Q mutant exhibited reduced membrane binding of [125I]TSH and impaired signal transduction in response to TSH. In contrast, the W546X mutant was nonfunctional, with negligible membrane radioligand binding. Our findings indicate that a single normal TSH receptor allele is sufficient for normal thyroid function, but that the compound abnormality in the proband leads to TSH resistance.     

Interleukin-7: a cofactor for V(D)J rearrangement of the T cell receptor beta gene

The diversity of the T cell receptor repertoire is generated by rearrangement of gene elements in immature thymocytes. To identify a thymic signal that induces this rearrangement, a variety of agents were tested for their ability to induce rearrangement of the T cell receptor beta gene in suspensions of thymocytes from mouse embryos at day 14 of gestation. Of 16 agents tested, only interleukin-7 (IL-7) induced V(D)J gene rearrangement and sustained expression of the RAG-1 and RAG-2 genes, which are known to control rearrangement. These data implicate IL-7, a cytokine that is abundantly expressed in embryonic thymus, in driving gene rearrangement during early T cell development.     

Mutation (677 C to T) in the methylenetetrahydrofolate reductase gene aggravates hyperhomocysteinemia in hemodialysis patients

Hyperhomocysteinemia is frequent in hemodialysis patients and represents an independent risk factor for vascular disease in these patients. Elevated total homocysteine (tHcy) plasma levels can results from defective remethylation of Hcy to methionine due to decreased activity of the enzyme methylenetetrahydrofolate reductase (MTHFR). A genetic aberration in the MTHFR gene (677 C to T substitution) has been shown to result in reduced MTHFR activity. We tested the hypothesis that elevation of tHcy plasma levels in hemodialysis patients is influenced by the 677 C to T mutation of the MTHFR gene and examined the relation of the genotype with tHcy, folate and vitamin B12 plasma levels in these patients. The allelic frequency of the MTHFR mutation was evaluated in 203 patients maintained on chronic hemodialysis treatment. Total Hcy, folate, vitamin B12 levels and the MTHFR mutation were analyzed in 69 of the 203 patients and in 69 age- and sex-matched healthy control subjects. The allelic frequency of the 677 C to T transition in the MTHFR gene in hemodialysis patients was 34.7% versus 35.5% in healthy controls. Of 203 patients 26 (12.8%) were homozygous for the mutation (+/+) versus 10.2% in healthy subjects. The heterozygous (+/-) genotype was identified in 43.8% of patients versus 50.7% in controls. The mean tHcy level in hemodialysis patients was 28.7 +/- 11.0 mumol/liter versus 10.0 +/- 3.0 mumol/liter in control subjects. The mean tHcy levels were 36.4 +/- 13.4 mumol/liter in (+/+) patients and 12.2 +/- 4.5 mumol/liter in (+/+) controls, 28.7 +/- 10.8 mumol/liter in (+/-) patients and 9.9 +/- 2.7 mumol/liter in (+/-) controls and 25.4 +/- 8.5 mumol/liter in (-/-) hemodialysis patients versus 9.7 +/- 2.8 mumol/liter in (-/-) controls: There was no significant difference of folate and vitamin B12 concentrations in patients and controls with different MTHFR genotypes. Analysis of covariance including age, gender, folate concentrations, vitamin B12 levels, albumin and creatinine as covariables revealed a significant influence of the (+/+) genotype, albumin and folate status on tHcy levels in hemodialysis patients. Together, our data demonstrate that the extent of hyperhomocysteinemia in hemodialysis patients is not only the result of uremia or folate status, but is also genetically determined by the (+/+) MTHFR genotype. The presence of the 677 C to T mutation in the MTHFR gene does not appear to represent a risk factor for development of end-stage renal disease.     

Extensive phenotypic analysis of a family with growth hormone (GH) deficiency caused by a mutation in the GH-releasing hormone receptor gene

We studied the effects of aerosolized as well as intravenous infusion of acetylcholine on bronchial blood flow in six anesthetized sheep. Intravenous infusion of acetylcholine, at a dose of 2 microg/kg, increased bronchial blood flow from 45 +/- 15 (SE) to 74 +/- 30 ml/min, and vascular conductance increased by 76 +/- 22%. In contrast, aerosolized acetylcholine at doses of 2 and 20 microg/kg decreased bronchial vascular conductance by approximately 10%. At an aerosolized dose of 200 microg/kg, the bronchial vascular conductance increased by approximately 15%, and there was no further increase in conductance when the aerosolized dose was increased to 2,000 microg/kg. Pretreatment of animals with a nitric oxide synthase inhibitor, Nomega-nitro-L-arginine methyl ester hydrochloride, partially blocked the vasodilatory effects of intravenous acetylcholine and completely blocked the vasodilatory effects of high-dose aerosolized acetylcholine. These data suggest that aerosolized acetylcholine does not readily penetrate the vascular wall of bronchial circulatory system and, therefore, has minimal vasodilatory effects on the bronchial vasculature.     

Identification of Hb C [beta 6(A3)Glu-->Lys] in a Thai male

The propositus was a 29-year-old Thai male, whose electrophoretic pattern showed Hb A (58%) plus an abnormal hemoglobin (42%) with mobility identical to Hb A2 and Hb E. Protein sequencer analysis and tryptic peptide mapping of the beta chain indicated that the abnormal hemoglobin was Hb C [beta 6(A3)Glu-->Lys], rather than Hb E which is more commonly found in South East Asia. This conclusion was confirmed by direct sequence analysis of the propositus' DNA, which showed AAG as well as GAG at codon 6 of the beta gene, in agreement with heterozygosity for Hb C and Hb A. Furthermore, the beta gene framework (Ava II-, Bam HI+) of the propositus suggested that the beta C gene may have arisen from an independent mutation. Since Hb C and Hb E have the same mutation (Glu-->Lys) in the beta chain, although at different positions, and behave similarly in electrophoresis, cases of Hb C and Hb E may sometimes have been mistakenly identified for each other, based on whichever variant is most prevalent in the particular population.     

Triiodothyronine binding to lymphocytes from euthyroid subjects and a patient with peripheral resistance to thyroid hormone

Triiodothyronine (T3) binding to Ficoll-Isopaque purified human lymphocytes was studied. During incubation of lymphocytes with [125I]T3 in a calcium-free medium at 37 degrees C, maximal uptake of T3 in nuclei occurrred after 2 h and declined after prolonged incubationd incubation . Incubation of lymphocytes with T3 concentrations ranging from 1 X 10(-11) TO 1 X 10(-9) mol/l and subsequent treatment with Triton X-100 to strip off [125I]T3 bound with low affinity was used for the estimation of affinity and capacity of nuclear T3 binding sites. The mean equilibrium affinity constant (Ka) estimated with the Scatchard method in 11 euthyroid healthy subjects was 4.5 X 10(9) l/mol, and the mean maximal binding capacity 25 X 10(-5) mol/100 mug DNA. In a female patient with peripheral resistance to thyroid hormone action, the estimated Ka was 3.5 X 10(9) l/mol and the number of T3 binding sites 37 X 10(-15) mol/100 mug DNA. Although not statistically different from the mean value in euthyroid subjects, this Ka value was outside the range of control values observed and was considered presumptive evidence that the nuclear T3 receptors in this patient have abnormally low affinity for its ligand. The nuclear T3 binding capacity in this patient was significantly increased.