Analysis of Tg transcripts by real-time RT-PCR in the blood of thyroid cancer patients

Serum Tg (sTg) assays are sometimes unsatisfactory for monitoring thyroid cancer because interference caused by anti-Tg antibodies may reduce the sensitivity of the tests during thyroid hormone therapy. We have therefore developed a complementary method using real-time quantitative RT-PCR based on the amplification of Tg mRNA. Two different pairs of primers were used for the determination of the frequency of one of the variants of the alternative splicing of Tg mRNA. The frequency of this variant was as high in patients (n = 40) as in controls (n = 30), accounting for about 33% of the total Tg mRNA. Using appropriate primers, we observed that Tg mRNA values in controls varied according to the volume of thyroid tissue and the TSH concentration. The Tg mRNA values allowed the definition of a positive cutoff point at 1 pg/microg total RNA. This cutoff point, tested on the group of patients treated for thyroid cancer, produced fewer false negative results than those obtained with sTg assays. The standardized, highly sensitive real-time RT-PCR technique may therefore prove useful as a complement to sTg assays, particularly for patients with recurrent thyroid cancer receiving T(4) therapy.     

分子信标实时荧光定量PCR构绵羊PrP基因质粒和标准曲线

目的为能够用实时荧光定量PCR研究消化系统PrPmRNA的表达水平,构建目的基因PrP的标准品质粒和标准曲线。方法根据GenBank中绵羊基因编码区保守序列,设计特异性引物和分子信标探针;提取总RNA,经反转录、RT—PCR后,提取质粒,PCR-SSCP及测序鉴定,获得ARQ质粒;将质粒梯度稀释,进行荧光定量PCR,获得标准曲线及回归方程。结果结果显示,本实验设计的分子信标具有特异性强、灵敏度高和结果准确的特性;标准曲线相关系数r。=0．999,表明线性关系好,成功构建了目的基因的标准品质粒和标准曲线。     

Stimulation of Candida albicans transition by human chorionic gonadotrophin and a bacterial protein.

Candida albicans, a dimorphic fungus, is involved commonly in human infections with the mycelium form more associated with pathogenicity. The influence of various hormones and a bacterial protein on the transition from blastospore to mycelium was assessed. Human luteinizing hormone (hLH), chorionic gonadotrophin (hCG), and an hCG-like material purified from a bacteria, Xanthomonas maltophilia (PCG), were able to increase the rate of transition when compared with the controls. The effect of the two hormones and the bacterial peptide were specific, as human follicle stimulating hormone (hFSH), thyroid stimulating hormone (hTSH), growth hormone (hGH), prolactin (hPrl) and rat and bovine LH (rLH, bLH), and bovine albumin and gamma globulin did not affect the transition. The binding of 125I-hCG or 125I-LH to spheroplasts of Candida albicans were competitively displaced by hCG, hLH, and PCG. Scatchard analysis of binding of all three ligands revealed two binding sites with a high-affinity nM Kd. Thus, hCG, hLH, and PCG induce transition of Candida albicans from a blastospore state to a mycelium form, suggesting that these hormones may modify the pathogenicity of Candida albicans.     

Early secretion of chorionic gonadotrophin by marmoset embryos in vivo and in vitro

The control mechanisms of early pregnancy in primates differ from those in non-primate species in the early secretion of chorionic gonadotrophin (CG) by the embryo and in the support of the corpus luteum. This study describes the initiation of secretion of CG by the embryo of the marmoset monkey examined in vivo and in vitro. A bioassay for gonadotrophin, which did not distinguish between CG and LH, was adapted and validated for the marmoset. A system of embryo culture was developed whereby embryos were grown from morula/blastocyst stages until at least the differentiation of the trophoblast and yolk sac, facilitated by the embryo attaching to a monolayer of marmoset fibroblast cells. Gonadotrophin concentrations were measured in the peripheral circulation of marmosets during precisely timed stages during the first 84 days of the 144-day gestation period, providing a profile of secretion that was maintained at high levels for longer than the profile seen in Old World primates, including man. A clear increase above baseline levels was seen by day 17 after ovulation, implantation commencing in the marmoset on days 11-13. Gonadotrophin was secreted by embryos in culture from the time of attachment in vitro, but there was no clear evidence of secretion before attachment. Levels of gonadotrophin secreted by embryos in vitro increased rapidly, reaching a maximum mean production rate of 90 mIU/24 h within 4 days after attachment. The experimental systems developed here will allow the examination of the local function of CG at the implantation site, intra-embryonic control of its secretion and its possible involvement in embryonic development.     

Precocious ovulation induced by human chorionic gonadotrophin in the immature female rat: comparison of follicle growth induced by treatment with human chorionic gonadotrophin and by electrical stimulation of the hypothalamus

Precocious first ovulation, preceded by an endogenous preovulatory LH surge, could be predictably induced in immature female rats by administering repeated injections of human chorionic gonadotrophin (hCG). Administration of a dose of 0.05-0.075 i.u. hCG, four times a day from day 28 to day 31 of age resulted in a highly constant ovulatory response: at 4.0 +/- 0.0 days after the start of treatment 7.7 +/- 0.3 (n = 15) ova were found. Use of a higher dose of hCG (0.1 i.u.) resulted in lower numbers of ova (5.6 +/- 0.4, n = 7; P less than 0.005) whereas use of a lower dose of hCG (0.025-0.038 i.u.) resulted in a less constant timing of the induced ovulation at 5.4 +/- 0.2 days after the start of treatment (n = 7; P less than 0.0005). In animals treated with the dose of 0.05-0.075 i.u. hCG, a positive correlation was found between body weight at the start of treatment and the number of ova released (r = 0.75, n = 25; P less than 0.001). Ovarian follicle dynamics were studied on the various days of hCG treatment (dose 0.05-0.075 i.u.) and compared with the follicle changes that take place after electrical stimulation of the hypothalamus, performed on day 28, a treatment known to result in first ovulation 4-5 days later. In both groups a decrease in the number of the smallest and the middle-sized antral follicles as compared with their respective controls was seen, whereas numbers of follicles in the largest, 'ovulatable' size classes gradually increased.(ABSTRACT TRUNCATED AT 250 WORDS)     

A novel form of ectopic human chorionic gonadotrophin beta-subunit in the serum of a woman with epidermoid cancer

A novel form of free human chorionic gonadotrophin beta-subunit (hCG beta) was found in serum from ElBre, a woman with epidermoid carcinoma of unknown origin. ElBre hCG beta was larger than standard (pregnancy urine) hCG beta when analysed by gel chromatography (apparent molecular weight 54 000 vs 44 000). This size difference appeared to be due to a larger carboxyterminal extension (CTE) of ElBre hCG beta since thermolysin cleavage of the CTE from standard hCG beta and Elbre hCG beta yielded core products of the same size. Oligosaccharides, O-linked to serine or threonine, were present in ElBre hCG beta, presumably on its CTE as judged by the complete binding of desialylated ElBre hCG beta to immobilized peanut agglutinin (this lectin is specific for terminal galactose linked beta 1----3 to N-acetylgalactosamine, a disaccharide exposed after desialylation of the O-linked oligosaccharides of standard hCG beta). ElBre hCG beta, however, was incompletely recognized by antisera specific for the CTE of standard hCG beta, especially the carbohydrate-sensitive antiserum R141. The O-linked oligosaccharides of standard hCG beta are heterogeneous in size; 13% are of the largest (hexasaccharide) form. In contrast, over 50% of the O-linked oligosaccharides in hCG beta from the JAr choriocarcinoma cell line are hexasaccharides. Like desialylated ElBre hCG beta, desialylated JAr hCG beta bound completely to peanut agglutinin, but was incompletely recognized by antisera to the hCG beta-CTE. Furthermore, JAr hCG beta was intermediate in size between standard hCG beta and ElBre hCG beta when analysed by gel chromatography (apparent molecular weight 49 000).(ABSTRACT TRUNCATED AT 250 WORDS)     

An international study to standardize the detection and quantitation of BCR-ABL transcripts from stabilized peripheral blood preparations by quantitative RT-PCR

Due to the lack of comparability of BCR-ABL mRNA quantification results generated by various methodologies in different laboratories, an international multicenter trial was started with the participation of six laboratories (platforms: LightCycler LC, n=3; TaqMan TM, n=3). One hundred and eighty-six PB samples derived from healthy donors were spiked with serial dilutions (1:20 to 1:2x10(6)) of b2a2, b3a2 or e1a2 BCR-ABL positive white blood cells (WBC) from leukemic patients. After PAXgene stabilization, blinding, freezing and distribution, standardized RNA extraction, cDNA synthesis, PCR protocols and data evaluation were carried out. There was no significant difference in the results achieved using LC and TM technologies, but a considerable overall variation (CV=0.74 for ratios BCR-ABL/ABL). Up to a dilution of 1:1,000, 27/30 of the 2.5 mL samples tested positive. For higher dilutions, a PB volume of 5 or 10 ml was required to improve sensitivity. The study showed the feasibility of RQ-PCR standardization independent of the PCR machine used.     

Co-operation of progesterone and prostaglandins in ovulation induced by human chorionic gonadotrophin in immature rats primed with pregnant mare serum gonadotrophin

Serial injections of a mixture of prostaglandin (PG) E2 and F2 alpha 0, 2, 4, and 6 h after simultaneous injection of human chorionic gonadotrophin (hCG) and indomethacin incompletely restored the ovulation that would have been blocked by indomethacin in immature rats treated with pregnant mare serum gonadotrophin followed by hCG. Serial injections of another mixture of PGE2 and PGF2 alpha 6, 8, 10 and 12 h after simultaneous injection of hCG and indomethacin similarly reversed, in part, the inhibitory effects of indomethacin on hCG-induced ovulation. In contrast, serial injections of the mixtures of PGE2 and PGF2 alpha 0, 2, 4, 6, 8, 10 and 12 h after simultaneous injection of hCG and indomethacin completely restored the indomethacin-blocked ovulation, suggesting that the prostaglandins mediate the action of hCG on ovulation both in the earlier and later stages of the preovulatory process. Six hours after simultaneous injection of hCG and indomethacin serial injections of a mixture of PGE2 and PGF2 alpha reproduced the acute and temporary increase in concentrations of progesterone and testosterone in plasma which would have been abolished by indomethacin. Progesterone given concurrently with hCG and indomethacin partially antagonized the inhibitory action of indomethacin on ovulation. Serial injections of a mixture of PGE2 and PGF2 alpha 6, 8, 10 and 12 h after concurrent administration of progesterone with hCG and indomethacin completely restored the indomethacin-blocked ovulation, suggesting that progesterone can substitute the action of prostaglandins injected serially in the first half of the preovulatory process. It was concluded that the co-operation of progesterone in the earlier stage and of prostaglandins in the later stage of the preovulatory interval is required to mediate the action of hCG on ovulation.     

Direct in-vivo detection of atypical hormonal expression of a Sertoli-Leydig cell tumour following stimulation with human chorionic gonadotrophin

A 60-year-old woman presented with progressive hirsutism and elevated serum testosterone levels. Selective bilateral ovarian and adrenal vein catheterization demonstrated mild elevated testosterone and androstenedione levels in the right ovarian vein, which increased considerably 15 minutes following intravenous injection of 5000 IU human chorionic gonadotrophin. Androgen levels decreased remarkably after administration of gonadotrophin hormone releasing hormone-agonist (GnRH-a). On histological examination, diffuse stromal hyperplasia of both ovaries was noted, with a small Sertoli-Leydig cell tumour in the right ovary. This is the first report of preoperative, direct selective diagnosis of a small Sertoli-Leydig cell tumour with such a hormonal expression. Ovarian Sertoli-Leydig cell tumours are rare sex cord stromal tumours that exhibit testicular-like structure and differentiation. These tumours are potentially malignant, can cause progressive virilization (Young & Scully, 1985), and are often clinically manifested as palpable pelvic masses and virilization (Meldrum & Abraham, 1979; Friedman et al., 1985). We describe a patient with postmenopausal virilization due to Sertoli-Leydig cell tumour, in whom a remarkable increase in androgens was detected following intravenous human chorionic gonadotrophin injection, during adrenal and ovarian selective vein blood sampling. A remarkable decrease in the serum androgen level was noticed following an injection of gonadotrophin hormone releasing hormone-agonist (GnRH-a).     

联合应用激光微切和实时定量RT-PCR检测单个胰岛的基因表达

在显微镜下用紫外激光微切机将单个胰岛从胰腺切片中切下，提取RNA后逆转录成cDNA并在实时定量RY—PCR中得到有效的扩增，其表达量与细胞数量成正比。     

Pathological and molecular aspects of prostate cancer

This review focuses on new findings and controversial issues in the the pathology and molecular biology of adenocarcinoma of the prostate. Since management of high-grade prostatic intraepithelial neoplasia on needle biopsy--the most common precursor lesion to prostate cancer--is the crucial issue with this lesion, we discuss the risk of cancer subsequent to this histological diagnosis and the issue of whether such neoplasia should be regarded as carcinoma-in-situ. We also look at prostate cancer itself, starting with its diagnosis, reporting on needle biopsy, and reviewing how the most frequently used grading system, the Gleason grading system, affects treatment. The molecular basis of prostate cancer includes inheritable and somatic genetic changes (tumour suppressor genes, loss of heterozygosity, gene targets and regions of chromosomal gain, CpG island promoter methylation, invasion and metastasis suppressor genes, telomere shortening, and genetic instability). Changed gene expression (eg, proliferation-related genes, changes in the androgen receptor, apoptosis and stress-response genes) have potential as biomarkers and therapeutic targets in prostate cancer.