Evidence that the thyroid-stimulating hormone (TSH) receptor transmembrane domain influences kinetics of TSH binding to the receptor ectodomain

Thyroid-stimulating hormone (TSH)-induced reduction in ligand binding affinity (negative cooperativity) requires TSH receptor (TSHR) homodimerization, the latter involving primarily the transmembrane domain (TMD) but with the extracellular domain (ECD) also contributing to this association. To test the role of the TMD in negative cooperativity, we studied the TSHR ECD tethered to the cell surface by a glycosylphosphatidylinositol (GPI) anchor that multimerizes despite the absence of the TMD. Using the infinite ligand dilution approach, we confirmed that TSH increased the rate of dissociation (k(off)) of prebound (125)I-TSH from CHO cells expressing the TSH holoreceptor. Such negative cooperativity did not occur with TSHR ECD-GPI-expressing cells. However, even in the absence of added TSH, (125)I-TSH dissociated much more rapidly from the TSHR ECD-GPI than from the TSH holoreceptor. This phenomenon, suggesting a lower TSH affinity for the former, was surprising because both the TSHR ECD and TSH holoreceptor contain the entire TSH-binding site, and the TSH binding affinities for both receptor forms should, theoretically, be identical. In ligand competition studies, we observed that the TSH binding affinity for the TSHR ECD-GPI was significantly lower than that for the TSH holoreceptor. Further evidence for a difference in ligand binding kinetics for the TSH holoreceptor and TSHR ECD-GPI was obtained upon comparison of the TSH K(d) values for these two receptor forms at 4 °C versus room temperature. Our data provide the first evidence that the wild-type TSHR TMD influences ligand binding affinity for the ECD, possibly by altering the conformation of the closely associated hinge region that contributes to the TSH-binding site.     

The histidin-loop is essential for transport activity of human MDR3. A novel mutation of MDR3 in a patient with progressive familial intrahepatic cholestasis type 3

Experimental evidence has been provided that a histidine-loop within the nucleotide binding domain of ABC transporter is essential for efficient function of this class of transporter proteins. Here we report the first patient with a mutation of the putative histidine-loop of a human ABC transporter, the multi drug resistance protein 3 (MDR3). The patient presented at the age of 4years with a history of severe pruritus, elevated serum gamma-glutamyltransferase and bile acid levels since several years suggesting the diagnosis of progressive familial intrahepatic cholestasis type 3 (PFIC-3) due to defects in MDR3. Liver biopsy demonstrated an apparently normal MDR3 expression, however, genetic analysis revealed a novel homozygous mutation in the ABCB4 gene (c.3691C>T) in the patient. This mutation was associated with a change of histidine to tyrosine at amino acid position 1231 of MDR3 (p.H1231Y). As shown by sequence alignment, this amino acid corresponds to the highly conserved histidine of the "H-loop", which is critical for ATP-hydrolysis, suggesting an essential role of histidine 1231 of human MDR3.     

TRH synthesis in “mute” thyrotropinomas: cause‐effect or coincidence?

In the pathogenesis of thyrotropin (TSH) immunopositive pituitary adenomas, trigger mutagenetic events are well recognized. However, the way towards a clinical significant tumor is followed under the pressure of growth factors, among which the intrapituitary synthesis of releasing factors could bring a significant contribution. In this study, the production of thyrotropin releasing hormone (TRH) and beta TSH chain was evaluated at the mRNA level by in situ hybridization and end product level by immunohistochemistry, in 18 patients submitted to neurosurgery for pituitary macroadenomas. The hormonal sampling showed abnormal secretion for FSH in 5 and TSH in 4 patients. Seven cases were immunopositive for TSH, and expressed TSH β mRNA. All but one out of these expressed also TRH mRNA. FSH immunoreactivity was documented in 12/ 18, only one of these being negative for TRH mRNA. Paracrine TRH could contribute to the patogenesis of these "mute" adenomas.     

Long-term EGF/serum-treated human thyrocytes mimic papillary thyroid carcinomas with regard to gene expression

Constitutive activation of the RAS/RAF/MAPK pathway has been found in different tumor types including papillary thyroid carcinomas (PTCs). To get more insight into genes primarily regulated in the human tumor cells, an in vitro model was developed in which primary cultures of human thyrocytes were treated for different times with epidermal growth factor and serum (EGF/serum), which stimulate the MAPK cascade. Gene expression profiles were obtained by microarrays and compared to the expression profiles of PTCs. An evolution from short-term to long-term EGF/serum-treated cells was found, i.e., a program change showing a distinction between gene expression profiles of short-term and long-term EGF/serum-treated cells. The late pattern of EGF/serum stimulated cells converges to the pattern of PTCs. Comparison of these two types of cells with cAMP activated cells, from thyroid-stimulating hormone-treated thyrocytes and autonomous adenomas, showed distinct gene expression profiles for the two pathways. For the two models, an overlap was found in a number of genes which were early induced in vitro but down-regulated later in vitro and in the in vivo tumors. Thus, long-term stimulated human primary cultures demonstrate a clear relation with the tumor in vivo and could therefore be used as models for the disease.     

Metabolic and endocrine effects of water and/or food deprivation in rats

Metabolic and endocrine effects of water and/or food deprivation in rats. We aim at studying the effect of water deprivation, food deprivation and their combination for three days on adrenal cortex, pituitary-thyroid axis and vasopressinergic system activity in rats. Corticosterone level was determined by fluorimetric method. The levels of free thyroxine (FT4) and thyroid stimulating hormone (TSH) were determined by immunoenzymatic assay and vasopressin (AVP) level was determined by radio-immunoassay. In all three groups, basal levels of plasma corticosterone were increased. A thyroid dysfunction was shown after water deprivation, food deprivation and their combination reflected by a significant decrease in FT4 levels. Paradoxically, a significant decrease in TSH level was observed in food-deprived rats and in rats subjected to simultaneous food and water deprivation, while a slight and not significant decrease in TSH level was shown in water-deprived rats. A significant increase in plasma AVP level was observed after water deprivation and simultaneous water and food deprivation, while no change was found after food deprivation. The data indicated that water deprivation, food deprivation and their combination stimulated the adrenal cortex, thereby suggesting a stress state. On the other hand, it seems that nutritional stress modifies the pituitary-thyroid axis through mechanisms different from those of osmotic stress. Moreover, it seems that food deprivation partially prevented the stimulatory effect of water deprivation on vasopressinergic system.     

Successful microdissection testicular sperm extraction for men with non-obstructive azoospermia

Non-obstructive azoospermia (NOA) is the most severe form of male infertility, defined by lack of spermatozoa in the ejaculate caused by impaired spermatogenesis. The chance of biological fatherhood of these men has been improved since the introduction of microdissection testicular sperm extraction (MD-TESE) combined with intracytoplasmic sperm injection. A thorough patient evaluation preoperatively is essential to recognize any underlying conditions, and to assist in patient counseling on the sperm recovery rate and pregnancy results. This review article summarizes the present data on MD-TESE to reach optimal results is treating men with NOA.     

促甲状腺激素对大鼠心肌成纤维细胞影响的研究

目的:探讨促甲状腺激素(TSH)对SD乳鼠心肌成纤维细胞(CFs)的影响及机制。方法:新生1-3 d的SD乳鼠15只,处死后进行CFs的分离与培养,倒置显微镜对原代培养细胞形态进行鉴定,免疫组织化学染色法对CFs胞浆内波动蛋白进行染色并鉴定。后续实验过程中,按照使用b TSH工作液的稀释浓度分为A组(1μmol·L~(-1))、B组(2μmol·L~(-1))、C组(4μmol·L~(-1))。MTT法检测心肌成纤维细胞增殖率。酶联免疫吸附法(ELISA)检测培养细胞的上清液中Ⅰ、Ⅲ型胶原蛋白含量。荧光定量PCR反应测定培养细胞上清液中基质金属蛋白酶-2(MMP-2)、基质金属蛋白酶-9(MMP-9)m RNA的相对表达量。Western-blot法检测培养细胞上清液中MMP-2、MMP-9的蛋白表达水平。结果:CFs的形态学变化及纯度鉴定结果显示乳鼠CFs培养成功。MTT试验结果显示,24 h时后,A、B、C组CFs的增殖率分别为112.91±10.23、123.22±9.34、132.56±9.36;48 h时后,A、B、C组CFs的增殖率分别为124.4±8.34、133.5±9.02、139.6±11.36。随着b TSH浓度的增加,CFs增殖率呈逐渐增加趋势。ELISA检测结果显示,A、B、C组I型胶原蛋白表达水平分别为(2.61±0.31)μg·L~(-1)、(5.53±0.66)μg·L~(-1)、(7.91±0.74)μg·L~(-1);A、B、C组Ⅲ型胶原蛋白表达水平分别为(3.96±0.25)μg·L~(-1)、(6.72±0.52)μg·L~(-1)、(8.11±0.65)μg·L~(-1)。随着b TSH浓度的升高,I型和Ⅲ型胶原蛋白表达水平呈升高趋势。荧光定量PCR法检测结果显示,A、B、C组MMP-2的m RNA表达量分别为9.124±1.021、15.223±1.536、18.678±1.742;A、B、C组MMP-9的m RNA表达量分别为16.447±1.664、17.402±1.881、24.247±2.364。随着b TSH浓度的增加,MMP-2、MMP-9的m RNA相对表达量呈增加趋势。Western-blot法检测结果显示,A、B、C组MMP-2灰度值分别为165.41±16.57、198.25±21.34、223.41±19.08;A、B、C组MMP-9灰度值分别为140.30±15.09、190.47±20.06、230.14±21.45。随着b TSH浓度的增加,MMP-2、MMP-9的蛋白表达水平呈增加趋势。结论:TSH能够促进乳鼠CFs细胞增殖及I、Ⅲ型胶原蛋白表达增加,导致细胞外基质成分降解及合成过程失调。