[Clinical effect and influencing factors of focused ultrasound ablation surgery combined with suction curettage for the treatment of mass-type cesarean scar pregnancy].

Objective: To investigate the clinical effect of focused ultrasound ablation surgery (FUAS) combined with suction curettage for mass-type cesarean scar pregnancy (CSP) and to analyze the influencing factors of vaginal bleeding and readmission. Methods: From January 2014 to December 2020, 88 patients with mass-type CSP were treated by FUAS combined with suction curettage in the Third Xiangya Hospital of Central South University. The clinical results and the influencing factors of bleeding and readmission for mass-type CSP were analyzed. Results: All the patients underwent one time FUAS treatment successfully. Immediately after FUAS treatment, color Doppler ultrasound showed obvious necrosis and no perfusion area in all lesions, and the blood flow in the mass-type CSP tissue significantly decreased. The median volume of blood loss in the procedure was 20 ml (range: 5-950 ml). Thirteen patients (15%, 13/88) had vaginal bleeding≥200 ml, and 15 patients (17%, 15/88) were hospitalized again. The average time for menstruation recovery was (28±8) days (range: 18-66 days). The average time needed for serum human chorionic gonadotropin-beta subunit to return to normal levels was (22±6) days (range: 7-59 days). The risk of large vaginal bleeding of patients were related to the blood supply of the mass (OR=5.280, 95%CI: 1.335-20.858, P=0.018) and the largest diameter of the mass (OR=1.060, 95%CI: 1.010-1.120, P=0.030). The risk of readmission were related to the largest diameter of the mass (OR=1.055, 95%CI: 1.005-1.108, P=0.030) and the depth of the uterus cavity (OR=1.583, 95%CI: 1.015-2.471, P=0.043). No serious complications such as intestinal and nerve injury occurred during and after FUAS treatment. Conclusions: FUAS combined with suction curettage is safe and effective in treating patients with mass-type CSP through this preliminary study. The volume of vaginal bleeding are associated with the blood supply of the mass and the largest diameter of the mass, the risk of readmission are related to the largest diameter of the mass and the depth of the uterus cavity.

Orbital-Selective Kondo Entanglement and Antiferromagnetic Order in USb_{2}.

In heavy-fermion compounds, the dual character of f electrons underlies their rich and often exotic properties like fragile heavy quasiparticles, a variety of magnetic orders and unconventional superconductivity. 5f-electron actinide materials provide a rich setting to elucidate the larger and outstanding issue of the competition between magnetic order and Kondo entanglement and, more generally, the interplay among different channels of interactions in correlated electron systems. Here, by using angle-resolved photoemission spectroscopy, we present the detailed electronic structure of USb_{2} and observe two different kinds of nearly flat bands in the antiferromagnetic state of USb_{2}. Polarization-dependent measurements show that these electronic states are derived from 5f orbitals with different characters; in addition, further temperature-dependent measurements reveal that one of them is driven by the Kondo correlations between the 5f electrons and conduction electrons, while the other reflects the dominant role of the magnetic order. Our results on the low-energy electronic excitations of USb_{2} implicate orbital selectivity as an important new ingredient for the competition between Kondo correlations and magnetic order and, by extension, in the rich landscape of quantum phases for strongly correlated f electron systems.

[Analysis of risk factors associated central venous catheter-related thrombosis in patients undergoing abdominal operation].

Objective: To analyze the risk factors of catheter-related right jugular venous thrombosis in patients undergoing abdominal surgery. Methods: A total of 125 patients (18-90 years, ASA Ⅰ-Ⅲ) scheduled for abdominal surgery underwent right internal jugular in our study. Ultrasound-guided central venous catheterization were carried out before surgery and Doppler ultrasound examination were conducted daily postoperatively until the catheter was removed. The patients were divided into thrombosis positive or negative group based on the Doppler ultrasound examinations. General clinical data of all patients including name, gender, age, BMI, preoperative Caprini score, bleeding, duration of operation and anesthesia, were also collected. D-dimer test was performed on the third postoperative day. Results: Of the 125 patients, 16(12.8%) were found to have catheter-related right internal jugular vein thrombosis. There were 16 cases (9 males and 7 females) in the thrombosis positive group, with an average age of (73±7) years old, body mass index (22.8±2.9) kg/m(2), and preoperative Caprini score (6.1±1.4).In the thrombosis negative group,among whom 72 were men and 37 were women. The average age of these patients was (66±11) years old, BMI was (22.6±2.9)kg/m(2), and preoperative Caprini score was (5.9±1.3).There was no statistical difference between the two groups regarding age, gender, BMI, preoperative Caprini score (P>0.05).The average operating time, anesthesia time, bleeding amount and D-dimer level on the third postoperative day were (189±46) min, (211±59) min, (288±96) ml,(3.4±1.6) mg/L in thethrombosis positive group, and (139±39) min, (171±46) min, (175±114) ml,(2.0±0.9) mg/L in the thrombosis negative group, respectively. Duration of surgery, bleeding amount and D-dimer level on the third postoperative day significantly affected the occurrence of catheter-related right internal jugular venous thrombosis (U=10.768, 359.000, 390.000, P<0.05), but no statistically significant differences in anesthesia time between the two groups.Logistic regression analysis screened out duration of surgery, bleeding amount and D-dimer level on the third postoperative day as risk factors for catheter-related right jugular venous thrombosis(OR=10.037, 1.011, 3.274, P<0.05). Conclusion: The high incidence of catheter-related right jugular venous thrombosis in patients undergoing abdominal surgery is closely related to intraoperative blood loss, operation time, D-dimer level on the third postoperative day and other factors.

[Clinical study of high intensity focused ultrasound ablation combined with GnRH-a and LNG-IUS for the treatment of adenomyosis].

Objective: To investigate the clinical effect of dysmenorrhea in patients with adenomyosis treated by high intensity focused ultrasound(HIFU)ablation combined with gonadotropin-releasing hormone agonist(GnRH-a)and levonorgestrel-releasing intrauterine system(LNG-IUS). Methods: From April 2012 to December 2015, 477 cases of adenomyosis patients with dysmenorrhea were treated by HIFU in the Third Xiangya Hospital. Among them, some patients were treated with HIFU alone, some of them were treated with HIFU combined with GnRH-a and(or)LNG-IUS, thus were classified as H group, H+G group, H+M group and H+G+M group. The improvements of clinical results were compared among the four groups and the influencing factors of HIFU treatment for adenomyosis were also analyzed. Results: During the follow-up period, the overall effective rates of the treatment decreased with time, 3 months 89.4%(345/386), 12 months 84.0%(221/263), 24 months 74.2%(98/132), and the overall recurrence rate was 12.9%(39/303). The significant difference in the curative at 3 months[H group 83.7%(170/203), H+M group 95.0%(95/100), H+G group 100.0%(43/43), H+G+M group 96.8%(30/31)], 12 months[H group 79.4%(123/155), H+M group 93.2%(69/74), H+G group 11/12, H+G+M group 15/17], and 24 months[H group 68.0%(51/75), H+M group 96.4%(27/28), H+G group 6/12, H+G+M group 15/15]after HIFU treatment and recurrence rate[H group 19.0%(29/153), H+M group 3.3%(3/90), H+G group 19.4%(6/31), H+G+M group 4.5%(1/22)]were observed among the four groups(P<0.05). Pairwise comparison further showed that, in 3 months after the treatment, the effect of H group was significantly lower than those of H+M group and H+G group(P= 0.003, P=0.005); in 12 months after the treatment, the effect of H group was significantly lower than that of H+M group(P=0.006); while in 24 months after treatment, the effect of H group was significantly lower than that of H+G+M group(P=0.005), and the effect of H+G group was lower than that of H+G+M group(P= 0.001); and the recurrence rate of H group was significantly higher than that of H+M group(P<0.008). In patients of group H, the effect of HIFU was related to uterine size, the effect of patients with large uterine volume was significantly higher than that of small volume of uterine of patients(P=0.017, OR=2.739, 95%CI: 1.200- 6.251); with increasing of age, the improvement of dysmenorrhea had a increasing trend(P<0.05). Conclusions: HIFU combined with GnRH-a and(or)LNG-IUS could improve the treatment effect in relief of dysmenorrhea. Based on our results, individual treatment protocol should be selected for different patients.

Distinct dysregulation of lipid metabolism by unliganded thyroid hormone receptor isoforms.

Thyroid hormone receptors (TRs) play critical roles in energy homeostasis. To understand the role of TRs in lipid homeostasis in vivo, we adopted the loss-of-function approach by creating knock-in mutant mice with targeted mutation in the TRalpha gene (TRalpha1PV mouse) or TRbeta gene (TRbetaPV mouse). The PV mutation, identified in a patient with resistance to thyroid hormone, exhibits potent dominant-negative activity. Here we show that in contrast to TRalpha1PV mouse, TRbetaPV mice exhibited no significant reduction in WAT but had significant increases in serum free fatty acids and total triglycerides. Moreover, the liver of TRbetaPV mice was markedly increased (33%) with excess lipid accumulation, but the liver mass of TRalpha1PV mouse was decreased (23%) with paucity of lipids. These results indicate that apo-TRbeta and apo-TRalpha1 exerted distinct abnormalities in lipid metabolism. Further biochemical analyses indicate that increased lipogenic enzyme expression, activated peroxisome proliferator-activated receptor gamma (Ppargamma) signaling, and decreased fatty acid beta-oxidation activity contributed to the adipogenic steatosis and lipid accumulation in the liver of TRbetaPV mice. In contrast, the expression of lipogenic enzymes and Ppargamma was decreased in the liver of TRalpha1PV mice. These results suggest that the regulation of genes critical for lipid metabolism by TRs in the liver is isoform dependent. These results indicate that apo-TRbeta and apo-TRalpha1 had different effects on lipid metabolism and that both TR isoforms contribute to the pathogenesis of lipid metabolism in hypothyroidism.

Though with constraints imposed by endosymbiosis, preferential attachment is still a plausible mechanism responsible for the evolution of the chloroplast metabolic network.

Chloroplasts evolved as a result of endosymbiosis, during which sophisticated mechanisms evolved to translocate nucleus-encoded plastid-targeted enzymes into the chloroplast to form the chloroplast metabolic network. Given the constraints and complexity of endosymbiosis, will preferential attachment still be a plausible mechanism for chloroplast metabolic network evolution? We answer this question by analysing the metabolic network properties of the chloroplast and a cyanobacterium, Synechococcus sp. WH8102 (syw). First, we found that enzymes related to more ancient pathways are more connected, and synthetases have the highest connectivity. Most of the enzymes shared by the two densest cores between the chloroplast and syw are synthetases. Second, the highly conserved functional modules mainly consist of highly connected enzymes. Finally, isozymes and enzymes from endosymbiotic gene transfer (EGT) were distributed mainly in conserved modules and showed higher connectivity than nonisozymes or non-EGT enzymes. These results suggest that even with severe evolutionary constraints imposed by endosymbiosis, preferential attachment is still a plausible mechanism responsible for the evolution of the chloroplast metabolic network. However, the current analysis may not completely differentiate whether the chloroplast network properties reflect the evolution of the chloroplast network through preferential attachment or has been inherited from its cyanobacterial ancestor. To fully differentiate these two possibilities, further analyses of the metabolic network structure properties of organisms at various intermediate evolutionary stages between cyanobacteria and the chloroplast are needed.

Modeling thyroid cancer in the mouse.

Thyroid carcinomas, the most common endocrine tumors in humans, have an increasing incidence in the U.S. and worldwide. There are four major types of thyroid cancers: papillary, follicular, anaplastic, and medullary carcinomas. In recent years, significant progress has been made in the identification of genetic alterations in thyroid carcinomas, particularly, papillary and medullary thyroid cancers. Mouse models of thyroid cancer are valuable tools in elucidating molecular genetic changes underlying thyroid carcinogenesis and in identifying potential molecular targets for therapeutic intervention. Representative mouse models of papillary, follicular, and medullary carcinomas are reviewed here with particular emphasis on those for follicular thyroid carcinomas. Challenges for further development in the modeling of thyroid cancer will also be discussed.

Oxidation and corrosion behaviors of Mg-based nanoparticles.

Various Mg-based nanoparticles were prepared by evaporating bulk magnesium in the atmospheres of Ar, Ar+N2 and CH4, respectively. The formations, phases, morphologies, thermal properties and corrosion behaviors of these kinds of nanocomposite particles were investigated by means of X-ray diffraction (XRD), high-resolution transmission electron microscopy (HRTEM), thermogravimetric-differential thermal analysis (TG/DTA) and potentiodynamic polarisation scan (PPS). It is shown that the Mg-based nanoparticles produced in Ar and Ar+N2 atmospheres have hexangular crystal habits with particles' sizes ranging from 50 to 400 nm, while the nanoparticles produced in CH4 atmosphere have amorphous carbon out layers with particles' sizes among 20-100 nm. TG/DTA results show that two-steps oxidation process can be confirmed for all samples, which may be attributed to the oxidations of out layer and core of nanoparticle. The CH4 atmosphere-prepared nanoparticles exhibit better corrosion resistance properties due to its peculiar carbon doping.

Aberrant expression of Rab1A and its prognostic significance in human colorectal cancer.

OBJECTIVE: Colorectal cancer (CRC) is one of the most common types of cancer worldwide. Emerging evidence has verified that Rab1A plays an oncogenic role in several human malignancies including breast cancer, lung cancer, and hepatocellular carcinomas. However, the clinical significance and prognostic impact of Rab1A in CRC is still unclear. PATIENTS AND METHODS: We initiated our investigation by immunohistochemistry and Western blot analysis to confirm Rab1A expression in CRC tissues. Meanwhile, the correlation of Rab1A expression and clinicopathologic features, as well as outcome in CRC patients, were retrospectively analyzed. RESULTS: In the issue, Rab1A is overexpressed in CRC tissues compared with matched noncancerous tissues. Meanwhile, high Rab1A expression was significantly associated with the TNM stage, lymph node metastasis, and peritoneal metastasis. In addition, multivariate analyses identified Rab1A expression and TNM stage as independent predictors for CRC patients. Moreover, Kaplan-Meier survival analysis showed that patients with high Rab1A expression had a significantly worse survival time than those with low Rab1A expression, which especially affected the survival in CRC patients with advanced stage. Spearman analysis suggested that there was a positive relationship between Rab1A expression and preoperative serum carcinoembryonic antigen (CEA) for CRC patients. CONCLUSIONS: These results suggested that Rab1A is an important diagnostic marker for CRC, and Rab1A can be used as a valuable biomarker for prognosis as well as peritoneal metastasis in CRC patients. Rab1A may prove to be clinically useful for developing a new therapeutic target of CRC treatment.

Comparison of hepatic elimination of different forms of cholecystokinin in dogs. Bioassay and radioimmunoassay comparisons of cholecystokinin-8-sulfate and -33-sulfate.

The influence of hepatic transit on the ability of exogenous cholecystokinin-8-sulfate and -33-sulfate (CCK-8 and CCK-33, respectively) to stimulate gallbladder contraction and exocrine pancreatic secretion, as well as on the peripheral plasma concentration of each agent, was evaluated in five conscious dogs with pancreatic and gallbladder fistulas and complete portacaval transposition. The gallbladder pressure increments after portal administration of CCK-8 (0.125, 0.25, 0.50, and 1.0 microgram/kg per h for 5 min) were diminished by 36, 45, 39 and 25%, respectively, in comparison with those obtained with systemic administration of identical doses of CCK-8 (P less than 0.05). In a subsequent experiment, the integrated pancreatic juice volume, bicarbonate, and protein secretion were diminished by 22, 32, and 48%, respectively, during a 30-min infusion of CCK-8 (0.10 micrograms/kg per h) into the portal venous system, in comparison with the results obtained with systemic administration of CCK-8 (P less than 0.05). In contrast, the gallbladder pressure and pancreatic exocrine secretory responses to portal administration of CCK-33 did not differ significantly (P greater than 0.05) from the results obtained with systemic administration of CCK-33. Radioimmunoassay for CCK-8 in plasma showed that the integrated CCK-8 value during portal administration was significantly lower (P less than 0.05) than it was during systemic administration. The results for CCK-33, however, did not vary, whether it was given by a systemic or portal route (P greater than 0.05). Thus, the present study demonstrates that CCK-8 is partially inactivated by the liver whereas CCK-33 is not, which suggests that CCK-3 in the circulation may play a significant role in the physiologic regulation of the gallbladder and exocrine pancreas.

The orphan nuclear receptor Ear-2 is a negative coregulator for thyroid hormone nuclear receptor function.

Thyroid hormone (T3) nuclear receptors (TR) are ligand-dependent transcription factors which regulate growth, differentiation, and development. One emerging hypothesis suggests that TR mediate these diverse effects via a large network of coregulators. Recently, we found that TR-mediated transcriptional responses varied in six cell lines derived from different tissues. We therefore used human TR subtype beta1 (TRbeta1) as bait to search for coregulators in human colon carcinoma RKO cells with a yeast two-hybrid system. RKO cells exhibited T3-dependent and -independent transcriptional activation. One of the three positive clones was identified as Ear-2, which is a distant member of the chick ovalbumin upstream promoter-transcription factors of the orphan nuclear receptor family. The physical interaction between Ear-2 and TRbeta1 was further confirmed by specific binding of Ear-2 to glutathione S-transferase-TRbeta1. In addition, Ear-2 was found to associate with TRbeta1 in cells. As a result of this physical interaction, binding of TRbeta1 to the T3 response elements was inhibited. Using reporter systems, we found that both the basal activation and the T3-dependent activation mediated by TRbeta1 were repressed by Ear-2 in CV1 cells. In RKO cells, however, the T3-independent transcriptional activity was more sensitive to the repression effect of Ear-2 than the T3-dependent transcriptional activity. The repression effect of Ear-2 was reversed by steroid hormone receptor coactivator 1. These results suggest that TR-mediated responses reflect a balance of corepressors and coactivators in cells. These findings further strengthen the hypothesis that the diverse activities of TR are achieved via a large network of coregulators that includes Ear-2.

DNMT3A-mediated down-regulation of microRNA-105 promotes gastric cancer cell proliferation.

OBJECTIVE: It has been well-established that microRNAs (miRNAs), a class of short non-coding RNA molecules, play an important role in the development of gastric cancer. In the present study, we focused on miR-105, a novel miRNA not previously linked to gastric cancer. PATIENTS AND METHODS: 36 paired surgically resected gastric cancer tissues and matched adjacent normal tissues were used to detect the expression of miR-105. AGS cells were used to overexpress or silence of miR-105 and to determine its effect on several tumorigenic properties. A cell proliferation enzyme-linked immunosorbent assay was used to analyze the incorporation of BrdU during DNA synthesis of AGS cells. Total cDNA from AGS cells was used to amplify the 3'-UTR of YY1 by PCR and luciferase activity was determined using the Dual-Luciferase Reporter Assay System RESULTS: We found that expression of miR-105 was reduced in gastric cancer tissues, compared with adjacent normal tissues, due to hypermethylation at its promoter region. Overexpression of miR-105 suppressed, whereas its inhibition promoted cell viability and proliferation. We further identified Yin Yang 1 (YY1) as a direct target of miR-105, by which miR-105 exerted its anti-proliferative role. Moreover, we found that DNMT3A was responsible for the down-regulation of miR-105 in gastric cancer cells. CONCLUSIONS: Our data demonstrate that miR-105 inhibits gastric cancer cell proliferation and progression, which might provide a therapeutical target for cancer therapy.

Overexpression of miR-98 inhibits cell invasion in glioma cell lines via downregulation of IKKε.

OBJECTIVE: MicroRNAs (miRNAs) function as negative regulators for the expression of genes involved in cancer metastasis. The aim of this study was to investigate the potential role of miR-98 in gliomas and validate its regulatory mechanism. PATIENTS AND METHODS: Cell viability assays are used to measure proliferation of cell. mRNA expression is measured by qRT-PCR. Western blot analysis is used to measure protein expression. RESULTS: Functional studies showed that miR-98 overexpression inhibited glioma migration and invasion, but had no effect on the cell viability. An enhanced green fluorescent protein reporter assay, quantitative RT-PCR, and a western blot analysis confirmed that miR-98 suppressed the expression of IκB kinase (IKKε) by directly targeting its 3'-untranslated region, also, the NF-κB p65 nuclear translocation and matrix metalloproteinase (MMP)-9 expression were significantly arrested in glioma cells treated with miR-98 mimics. Accordingly, the overexpression of IKKε or NF-κB p65 can restore cell migration and invasion after being inhibited by miR-98, and can restore NF-κB p65 nuclear translocation as well as increase MMP-9 expression. CONCLUSIONS: These findings demonstrated that miR-98 functions as a tumor suppressor in gliomas. Furthermore, miR-98 may act as a potential therapeutic biomarker for glioma patients.

Differential sensitivity of thyroid hormone receptor isoform homodimers and mutant heterodimers to hormone-induced dissociation from deoxyribonucleic acid: its role in dominant negative action.

General resistance to thyroid hormone is an inheritable disease with resistance of peripheral tissues to elevated levels of thyroid hormone. Genetic studies have shown that it is due to interference in the functions of wild-type thyroid hormone nuclear receptors (wTRs) via the dominant negative effect of mutant TRs (mTRs). The present study compared the heterodimerization of the two TR isoforms, TR beta1 and TR alpha1, with mutant TRs to understand if mTRs had isoform-dependent dominant negative action. Using electrophoresis gel mobility shift assay, we have demonstrated that mutant PV, S, ED, and OK form heterodimers with wTR alpha1 and deltaTR beta1 (in which the A/B domain of wTR beta1 has been deleted), on the F2-thyroid hormone response element (TRE). In the presence of T3, both homo- and heterodimer complexes are dissociated in a T3 concentration dependent manner. The ED50 for deltaTR beta1 homodimers was 3-fold higher than that of wTR alpha1 homodimers. ED50s for deltaTR beta1/mTR heterodimers were 10- to 40-fold higher than the corresponding wTR alpha1/mTR heterodimers. Mutant ED and OK homodimers were only partially dissociated at the highest T3 concentrations used (100 nM), whereas no dissociation could be detected for PV and S homodimers, indicating differential sensitivity of the F2-bound TR dimers to the T3-induced dissociation. We presented a model that indicates the dissociation of any particular TR dimer from F2 is determined by competition of T3 for both of its constituent TRs. By transfection assays, we showed that the potency of the dominant negative action of PV on TR alpha1 and TR beta1 inversely correlated with the sensitivity of the appropriate mTR/wTR heterodimer to T3-induced dissociation from F2. The differential dominant negative action of mutants on the two TR isoforms could play an important role in the heterogeneity of tissue-specific manifestations in patients with resistance to thyroid hormone.

Understanding the molecular mechanism of dominant negative action of mutant thyroid hormone beta 1-receptors: the important role of the wild-type/mutant receptor heterodimer.

The clinical manifestations of patients with resistance to thyroid hormone result from inhibition of the functions of wild-type thyroid hormone receptors (wTRs) by the dominant negative effect of mutant TR beta 1 receptors (mTR beta 1). One of the proposed mechanisms by which mTR beta 1 exerts its dominant negative action is via formation of the putative inactive wTR beta 1/mTR beta 1 heterodimer. However, the nature of the wTR beta 1/mTR beta 1 heterodimer is poorly understood. The present study characterizes the wTR beta 1/mTR beta 1 heterodimer by electrophoretic mobility shift assay. The mutant TR beta 1 used was PV, which contains a frame shift mutation in the C-terminal part of TR beta 1 and has less than 1% of the T3 binding affinity of the wTR beta 1. Because of the difficulty in resolving wTR beta 1 and mutant PV dimers, we used a truncated wTR beta 1 in which the A/B domain was deleted (delta TR beta 1) to demonstrate the formation of the heterodimer on thyroid hormone response elements (TREs) in which the half-site binding motifs are oriented in an inverted repeat (F2), a direct repeat separated by four nucleotides (DR4), or an inverted repeat (Pal). Deletion of the A/B domain had no effect on the binding of T3 and TREs to wTR beta 1. In the presence of equal amounts of delta TR beta 1 and PV, three types of molecular complexes. delta TR beta 1 homodimer, delta TR beta 1/PV heterodimer, and PV homodimer bound to each TRE in a ratio of approximately 1:2:1. The identities of these complexes were confirmed by their ability to be supershifted by anti-TR beta 1 and/or anti-PV antibodies. delta TR beta 1/PV heterodimer formation varied with different TREs. The ratio of apparent affinity constant (Ka) in the binding of delta TR beta 1/PV to TREs was F2:DR4:Pal = approximately 6:2:1. The effect of T3 on delta TR beta 1/PV heterodimer formation was TRE dependent. No T3-induced dissociation was observed for the delta TR beta 1/PV heterodimer when bound to F2 and Pal. In contrast, the delta TR beta 1/PV heterodimer bound to DR4 was dissociated by T3 with an ED50 of 3.9 +/- 0.9 nM. The T3-induced dissociation of delta TR beta 1 homodimer bound to F2, DR4, and Pal had ED50 values of 4.1 +/- 1.2, 1.3 +/- 0.3, and more than 100 nM, respectively. By transfection assays, the dominant negative action of PV was found to be TRE dependent with the rank order of F2 >> Pal > ME (a DR4-like TRE in the rat malic enzyme gene). Taken together, these results indicate a strong correlation between wTR beta 1/mTR beta 1 heterodimer formation and the dominant negative potency of PV. These results suggest that the wTR beta 1/mTR beta 1 heterodimer could play an important role in the dominant negative action of mTR beta 1.

Intracerebroventricular administration of cholecystokinin-8 elevates plasma pancreatic polypeptide levels in awake dogs.

The effect of intracerebroventricular (ICV) administration of cholecystokinin-8 (CCK-8) (0.5, 1.0, 2.0, 4.0 micrograms) on plasma levels of pancreatic polypeptide (PP) was studied in conscious dogs. ICV administration of CCK-8 (1.0, 2.0, 4.0 micrograms) produced a rapid and transient elevation in plasma concentrations of PP. Peripheral muscarinic receptor blockade with atropine or truncal vagotomy abolished PP secretion induced by ICV CCK-8. Pretreatment with ICV atropine also prevented the elevation in plasma PP induced by CCK-8. Plasma levels of CCK-33/39, CCK-8, and gastrin were not affected by ICV CCK-8. Our results indicate that central administration of CCK-8 selectively elevated plasma PP concentration. Since the effect of CCK-8 on plasma PP was abolished by central and peripheral atropine pretreatment, as well as by vagotomy, central and peripheral vagal cholinergic mechanisms appear to participate in release of PP that is induced by CCK-8 given by ICV injection.

Dominant negative activity of mutant thyroid hormone alpha1 receptors from patients with hepatocellular carcinoma.

Complementary DNAs for two mutant thyroid hormone alpha1 receptors (TR alpha1) were isolated from hepatocellular carcinomas of two patients. Sequence analyses of the complementary DNAs showed a single Val390Ala and double Pro398Ser/Glu350Lys mutations in mutants H and L, respectively. We characterized their hormone-binding, DNA-binding, and dominant negative activities. Mutants H and L did not bind the hormone T3. Their DNA-binding activities were analyzed using three types of thyroid hormone response elements (TREs) in which the half-site binding motifs are arranged in an everted repeat (Lys), an inverted repeat (Pal), or a direct repeat separated by four nucleotides (DR4). Compared with wild-type TR alpha1 (w-TR alpha1), which bound these TREs with different homodimer/monomer ratios, binding of mutant L to the three TREs as homodimers was reduced by approximately 90%. However, binding of mutant H to these TREs was more complex. Although it bound normally to DR4 as homodimers, its binding to Lys as homodimers was reduced by approximately 80%. Surprisingly, its binding to Pal was markedly enhanced compared with w-TR alpha1. The binding of these two mutants to the three TREs as heterodimers with retinoid X receptors (RXR alpha and -beta) was not significantly affected. Consistent with the lack of T3-binding activity, both mutants had lost their trans-activation capacity. Mutants H and L exhibited dominant negative activity, but differed in their TRE dependency. The dominant negative potency of mutant H was in the rank order of Pal > DR4 > Lys, whereas no TRE dependency was observed for mutant L. The present study indicates that mutations of the TR alpha gene do occur in patients and that these novel TR alpha1 mutants provide a valuable tool to further understand the molecular basis of the dominant negative action of mutant TRs.

Identification of naturally occurring dominant negative mutants of thyroid hormone alpha 1 and beta 1 receptors in a human hepatocellular carcinoma cell line.

To understand the function of thyroid hormone nuclear receptors (TRs) in human hepatocellular carcinoma cells (HCC), we characterized the hormone binding and transactivational activity of TRs in a HCC cell line, J7. TR alpha 1 (J7-TR alpha 1) and TR beta 1 (J7-TR beta 1) complementary DNAs were cloned from this cell line, and the binding activity to the hormone response elements (TREs) and to the thyroid hormone, 3,3',5-triiodo-L-thyronine (T3) of the expressed TR proteins were evaluated. J7-TR alpha 1 and J7-TR beta 1 bound to TREs similarly as the TRs isolated from other tissues. However, J7-TR alpha 1 did not bind to T3, and J7-TR beta 1 bound to T3 with only about 10% the affinity of the wild-type TR beta 1. Sequencing of the complementary DNAs shows a single Met259Ile mutation in J7-TR alpha 1 and Met334Val in J7-TR beta 1. Using reporters containing TREs, we found that J7-TR alpha 1 and J7-TR beta 1 had virtually lost their transactivational activity. Moreover, these two mutants inhibited the transactivational activity of the wild-type TRs by a dominant negative effect not only on the transfected TRs, but also on endogenous TRs in other two HCC cell lines, SK-Hep-1 and HepG2. The potency of the dominant negative effect of these two mutants inversely correlated with the expression level of endogenous TRs. The present studies identified two novel naturally occurring TR mutants that have potent dominant negative action. The identification of both the alpha and beta dominant negative mutants in J7 made this cell line a useful model system to further understand the molecular mechanism of the dominant negative action of TR mutants.

Genetic classification of combined hepatocellular-cholangiocarcinoma.

Combined hepatocellular-cholangiocarcinoma (combined HCC/ CC) is a rare form of liver neoplasms showing both hepatocellular (HCC) and bile duct differentiation (CC). In an attempt to clarify the clonality and genetic/phenotypic relationships in the evolution of these neoplasms, we microdissected multiple HCC and CC foci and studied allelic status of chromosome arms 1p, 1q, 3p, 4q, 5q, 6q, 8p, 9p, 10q, 11q, 13q, 16q, 17p, 17q, 18q, and 22q. Overall, the highest frequency of loss of heterozygosity (LOH) was seen on 4q and 17p, followed by 8p and 16q. Of the 11 cases studied, 3 cases did not show any of the identical allelic losses between HCC and CC foci, indicating the biclonal nature. The remaining 8 cases showed multiple allelic losses shared between both components, strongly suggestive of a single clonal derivation. Moreover, 4 of the 8 cases showed additional or divergent allelic losses at more than 1 chromosomal locus only in HCC and/or CC foci. Thus, this heterogeneity was shown to affect the phenotypic diversity of the tumor. Summarizing the genetic patterns, combined HCC/CC could be classified into the following 3 possibilities: (1) collision tumor in which 2 independent neoplastic clones develop at close proximity; (2) single clonal tumor with homogeneous genetic background in both components--histological diversity is thus a manifestation of divergent differentiation potential of a single clone; (3) single clonal process in which genetic heterogeneity in the process of clonal evolution within the tumor parallels histologic diversity; therefore, the tumor in this category is mainly composed of mosaics of closely related subclones.

Expression of the mutant thyroid hormone receptor PV in the pituitary of transgenic mice leads to weight reduction.

Resistance to thyroid hormone (RTH) is a genetic disease caused by mutations of the thyroid hormone receptor beta gene (TRbeta). One of the symptoms in some affected individuals is growth retardation. To understand the molecular basis of growth retardation in these patients with RTH, a transgenic mouse was prepared in which the expression of the TRbeta1 mutant PV was targeted to the pituitary using the promoter of the glycoprotein hormone alpha-subunit. The PV mutant was originally identified in a patient with severe growth impairment. The PV mutation is a C-insertion at codon 448 of the TRbeta gene and leads to a frame-shift of the carboxyl-terminal 14 amino acids of TRbeta1, resulting in total loss of triiodothyronine (T3) binding and transcriptional activation. PV was selectively expressed in the pituitary of the transgenic mouse and not in other tissues examined. The transgenic mice showed a significant impairment in weight gain. However, no changes in the serum level of thyroid-stimulating hormone were seen, and no elevation of thyroid hormones was detected in the transgenic mice. The circulating levels of growth hormone and insulin-like growth factor I were not affected in the transgenic mice, suggesting that the growth impairment in RTH is complex and is mediated by pathways that are yet to be elucidated.