Β-subunit Of Human Chorionic Gonadotropin Research Articles

Evaluation of neoadjuvant therapy in patients with nongerminomatous malignant germ cell tumors

The authors evaluated the effectiveness of a neoadjuvant therapy (NAT) consisting of combined chemo and radiotherapy followed by complete resection of the residual tumor in patients with nongerminomatous malignant germ cell tumors (NGMGCTs). The authors treated 14 consecutive patients in whom NGMGCTs were diagnosed based on elevated levels of the tumor markers α-fetoprotein, human chorionic gonadotropin, and the β-subunit of human chorionic gonadotropin (β-HCG). Chemotherapy and radiotherapy were performed, and after the serum tumor markers level was in the normal or near-normal range, the residual tumors were completely resected. Residual tumors were confirmed in 11 of the 14 patients after NAT, and total removal was successful in 10 of the 11 patients. In the other patient the residual tumor could not be completely excised because it was attached to a deep vein. The follow-up duration ranged from 1.2 to 22.2 years. The 5-year event-free and total survival rates were 86% and 93%, respectively. Although 3 patients died, 2 of tumor recurrence and 1 of a radiation-induced secondary tumor (glioblastoma), the other 11 are alive and without evidence of tumor recurrence. The authors consider their NAT protocol for NGMGCT to be highly effective in relation to survival for the patients with NGMGCT, but there are several quality of life issues that need to be resolved.

Designing a Long-Acting Human Growth Hormone (hGH) by Fusing the Carboxyl-Terminal Peptide of Human Chorionic Gonadotropin β-Subunit to the Coding Sequence of hGH

Chimeric genes were constructed by fusing of human GH (hGH) cDNA to one, two, or three cassettes of the carboxyl-terminal peptide (CTP) of human chorionic gonadotropin (hCG)-beta-subunit. hGH variant genes were inserted into the pCI-DHFR plasmid, transfected into DG44 cells, and stable clones were selected. Bioactivity and pharmacokinetic studies were performed in hypophysectomized Sprague Dawley derived male rats. The results indicated that sc injections of GH-wild-type (WT), Biotropin (commercial), GH-CTP, or CTP-GH (0.6 mg/kg) once every 5 d for 11 d (total dose of 1.2 mg/kg) resulted in an increased weight gain by 4, 4.9, 5.1, and 7 g, respectively. Treatment with CTP-GH-CTP-CTP (GH-LA) or CTP-GH-CTP (0.6 mg/kg) once every 5 d for 11 d or with Biotropin (0.12 mg/kg) daily for 11 d (total dose 1.2 mg/kg) resulted in a dramatic increase in weight gain of 16.5, 16.8, and 17 g, respectively. Repeated injections with different doses of GH-LA, 0.6, 1.8 mg/kg every 4 d or daily injection of 0.12 mg/kg of Biotropin increased the weight gain by 16, 28, and 18 gr, respectively. In addition, the cumulative serum levels of IGF-I after injection of GH-LA was significantly higher than that detected after injection of Biotropin. Pharmacokinetic studies indicated that the half-life, mean residence time, area under the curve, time of maximal plasma concentration, and maximal plasma concentration of GH-LA are dramatically increased compared with Biotropin. This may suggest that the mechanism of GH metabolic clearance is affected by the presence of CTP. These data establish a rationale for using this chimera as a long-acting GH analog.

Gastric Cancer: Tumor Markers as Predictive Factors for Preoperative Staging

CEA, CA 19-9, and CA 72-4 are tumor markers commonly used for gastric neoplasms. The clinical importance of the preoperative serum levels of these tumor markers in gastric cancer (GC) is not well known. Even less is known about the predictive value of the preoperative serum levels of the β-subunit of human chorionic gonadotropin (hCGβ). We designed a prospective study to evaluate the significance of the preoperative values of these tumor markers in GC. The serum levels that we considered as positive are as follows: CA 72-4, >4 U/ml; CEA, >5ng/ml; CA 19-9, >37 U/ml; hCGβ-free subunit, <5 mUI/ml. These levels were correlated by pathological stage, lymph node status, and histology. We studied 66 (42 male and 24 female) patients prospectively. Twenty-seven patients had stage I and stage II GCs, while 39 patients had stage III and stage IV GCs. Two patients tested positive for hCGβ. The preoperative positivity rates of CA 72-4 in patients at the early stages (stages I and II) and in patients with advanced disease (stages III and IV) were 0 and 28 patients, respectively. The preoperative positivity rates of CEA/CA 19.9 were 0/5 and 7/12 patients in early stages and advanced disease, respectively. The serum levels of these markers were not correlated with the histological type or tumoral grade of GC. The preoperative serum level of CA 72-4 has the best predictive value in indicating advanced disease in patients diagnosed with GC. A combination of these four markers is better in predicting this situation.

Absorption, Distribution, Metabolism and Excretion of Corifollitropin Alfa, a Recombinant Hormone with a Sustained Follicle-Stimulating Activity

The follicle-stimulating hormone (FSH) analog corifollitropin alfa (Org 36286) is a potent FSH receptor agonist with an extended plasma half-life due to the fusion of the carboxy-terminal peptide (CTP) of the human chorionic gonadotropin β-subunit and the human FSH β-subunit. The absorption, tissue distribution, metabolism and excretion of corifollitropin alfa were studied in rats following a single subcutaneous administration of [¹²⁵I]corifollitropin alfa. The biological activity of [¹²⁵I]corifollitropin alfa was confirmed by an in vitro FSH receptor transactivation assay. Radioactivity in blood, serum, tissues and excreta was determined by radiometry up to 168 h post dosage. A drug-specific distribution occurred mainly to ovaries and the renal system. The distribution was similar in albino and pigmented rats, ruling out effects of melanin binding on distribution. Metabolites were studied in urine and serum by SDS-PAGE. The maximum concentration of 12 h indicated a slow absorption and excretion. Radioactivity was mainly (86%) excreted via urine. 90% of the radioactivity in serum was identified as [¹²⁵I]corifollitropin alfa, whereas only 7–15% of the radioactivity in urine was identified as [¹²⁵I]corifollitropin alfa and its dissociation products, the α- and β-subunits (including its CTP part). The remainder of the radioactivity in either matrix represented low-molecular-weight compounds resulting from catabolism and deiodination. In conclusion, the metabolic fate of corifollitropin alfa strongly resembles that of endogenous glycoprotein hormones, which predominantly consists of kidney clearance and the urinary excretion of the intact protein in parallel to kidney catabolism.

The fusion protein of HSP65 with tandem repeats of β-hCG acting as a potent tumor vaccine in suppressing hepatocarcinoma

It has been demonstrated that the β-subunit of human chorionic gonadotropin (β-hCG) is ectopically expressed on a variety of human cancers of different histological types and has been used as an antigenic target in anti-cancer vaccines. We engineered a fusion protein by fusing 10 tandemly repeated copies of the 10-residue sequence of β-hCG (109–118) (in CTP37) combined with β-hCG C-terminal 37 peptides to mycobacterial heat-shock protein 65 and immunized mice via subcutaneous injection. Humoral immune and cellular immune responses were effectively elicited. High titer of anti-β-hCG antibody was detected in immunized mice sera by ELISA and verified by Western blot analyses. The fusion protein of HSP65-X10-β-hCGCTP37 effectively inhibited the growth of tumor both protective and therapeutic anti-tumor immunity in hepatocellular carcinoma tumor models in mice. Meanwhile, it also attenuated tumor-induced angiogenesis in intradermal tumor model in mice. Taken together, these results demonstrate that immune responses are effectively induced by a novel fusion protein vaccine targeting β-hCG, suppressing the growth of hepatocellular carcinoma in mice. The β-hCG-targeted vaccine holds promise for the treatment of a number of cancers and merits further study.

Thyreotropic effect of human Chorionic Gonadotropin (hCG) in mares at estrus

The β-subunits of thyroid stimulating hormone (TSH) and human chorionic gonadotropin (hCG) share a high homology, just like the ectodomains of their receptors, do. As a consequence, hCG was shown to exert a thyrotropic action in humans and hamsters. This study aimed to investigate whether hCG, used to induce ovulation, displays a thyrotropic effect in the equine species too. Forty mares at estrus were divided in two groups; 20 were intravenously treated with sterile saline solution (NaCl 0.9%) (group A); 20 were intravenously treated with 4000 I.U. of hCG (group B). All the mares were artificially inseminated 24 hours after the administration of the drugs and underwent blood collection at estrus detection (T1), 2, 6, 24 hours later (T2, T3, T4), and 3 and 6 days later (T5, T6), in order to detect serum fT3 and fT4 concentrations. fT3 concentrations gave rise to similar and constant trends in the two groups, without any statistically significant difference, whereas fT4 concentrations declined in both groups, remaining statistically higher in group B than in group A for 48 hours (p < 0.05), thus accounting for a thyrotropic effect of hCG. Pregnancy rate was 70% in group B and 40% in group A, respectively. The higher success in the outcome of pregnancy may result from the immunomodulating and steroidogenic effects of hCG and from the higher levels of thyroid hormones observed in the hCG treated group.

Search for peptide immunogens of the beta-subunit of human chorionic gonadotropin (hCG) capable of eliciting hormone specific and neutralizing antisera. Identification of an undecapeptide eliciting hCG-specific antisera.

The work reported herein describes our attempts to identify peptide immunogens of the beta-subunit of the pregnancy-specific placental hormone, human chorionic gonadotropin (hCG), capable of eliciting hormone specific and neutralizing antisera. Hydrophilicity profiles of the beta-subunits of hCG and the homologous pituitary hormone, human luteinizing hormone (hLH) were compared and two sequences which are hydrophilic but unique to hCG-beta were identified. They are 6-12 and 109-119 of hCG-beta. Results of the studies with the undecapeptide 109-119 of hCG-beta are reported in this paper. The undecapeptide amide was synthesized using the p-(acyloxy) benzhydrylamine resin and antisera to the peptide were elicited in rabbits using the peptide-diphtheria toxoid conjugate. The peptide is highly immunogenic as both the rabbits responded with high titers of antibodies to the peptide. The antipeptide antibodies bound to hCG but not to hLH showing thereby that the region 109-119 of hCG-beta is a unique determinant of hCG. However, the antibodies were found not to neutralize the biological activity of hCG.

Semen quality in men with disseminated testicular cancer: relation with human chorionic gonadotropin β-subunit and pituitary gonadal hormones

To compare the semen quality and hormonal status between patients with testicular cancer and normal versus increased serum levels of beta-hCG. Retrospective study. Academic research environment. All 203 patients with testicular cancer who required chemotherapy in the period 1995-2003 were included. In 107 patients semen samples were stored by cryopreservation; 62 patients could be analyzed because both semen was stored and hormones were determined before starting chemotherapy (median age 25 years, range 17-49 years). Total motile sperm count, T, E(2), LH, FSH, and PRL. Total motile sperm count was decreased in patients with increased beta-hCG (median 11.9 x 10(6)) compared with patients with normal beta-hCG (median 21.5 x 10(6)). Testosterone, E(2), and PRL were significantly higher in patients with increased beta-hCG levels, whereas LH and FSH were lower. Semen quality was significantly and negatively correlated with beta-hCG, E(2), and PRL. Patients with increased beta-hCG had an inferior spermatogenesis compared with patients with normal beta-hCG. Increased beta-hCG appears to be associated with impaired spermatogenesis and increased levels of E(2) and PRL.

Inhibition of tumor growth in vitro and in vivo by a monoclonal antibody against human chorionic gonadotropin β

Human chorionic gonadotropin (hCG) β-subunit (hCGβ) has been detected in a wide variety of tumors and implicated in tumor maintenance and progression. To better facilitate the investigation of the expression and biological roles of hCGβ, we generated a set of monoclonal antibodies (mAbs) against hCGβ by the approach of DNA immunization. All the generated mAbs worked well in detecting native hCGβ antigen, while some of them were surprisingly found to exhibit potential cytotoxicity to tumor cells in our preliminary experiments. Here, one of those cytotoxic anti-hCGβ mAb 6H1 was evaluated in detail for its anti-tumor efficacy in vitro and in vivo. 6H1 showed high binding specificity to hCGβ, which was analyzed by Western blot and ELISA as well as indirect immunofluorescence assay. Treatment with 6H1 inhibited the growth of a panel of hCGβ-expressing tumor cell lines (HeLa, HL-60, HepG2, SMMC-7721, PC-3) in vitro. Moreover, 6H1 significantly delayed the growth of HeLa-borne tumors in nude mice and prolonged the survival of tumor-bearing mice. The anti-tumor effect of 6H1 was associated with the induction of apoptosis, which was estimated by Hoechst 33258 staining, DNA ladder assay and flow cytometry. Collectively, 6H1 revealed potent anti-tumor activity in vitro and in vivo and therefore may be an effective therapeutic candidate for immuno-intervention of cancers that ectopically express hCGβ.

Development of a Long-Acting Erythropoietin by Fusing the Carboxyl-Terminal Peptide of Human Chorionic Gonadotropin β-Subunit to the Coding Sequence of Human Erythropoietin

Human erythropoietin (EPO) is a glycoprotein hormone secreted from the kidney and controls red blood cell production. EPO has a wide clinical use in the treatment of anemia associated with renal disease, certain chronic diseases, and anemia related to chemotherapy and radiotherapy. One major issue regarding the clinical use of EPO is its relatively short half-life due to its clearance by glomerular filtration. Thus, the therapeutic protocol used in the treatment of patient-required frequent injections of EPO. To address this issue, we constructed a chimeric gene that contains the sequence of the carboxyl-terminal peptide (CTP) of human chorionic gonadotropin-beta subunit bearing four O-linked oligosaccharide recognition sites and the coding sequence of human EPO cDNA. Fusing the CTP to the carboxyl-terminal of EPO did not affect secretion, receptor binding affinity, or in vitro bioactivity. However, both in vivo potency and half-life of EPO-CTP were significantly enhanced. A single injection dose (660 IU/kg) of EPO wild-type administered once a week had no significant effect on haematocrit levels. However, EPO-CTP administered as 660 IU/kg once a week was effective as well as the same total dose of EPO wild-type administered as 220 IU/kg three times a week. This may emphasize the importance of sustained blood levels rather than total dose of administration for in vivo bioactivity. These data established the rationale for using this chimera as a long-acting EPO analog. The therapeutic efficacy of EPO-CTP analog needs to be established in higher animals and human clinical trials.

Human Chorionic Gonadotropin (hCG) Up-Regulates wnt5b and wnt7b in the Mammary Gland, and hCGβ Transgenic Female Mice Present with Mammary Gland Tumors Exhibiting Characteristics of the Wnt/β-Catenin Pathway Activation

Transgenic (TG) mice expressing human chorionic gonadotropin (hCG) beta-subunit under the ubiquitin C promoter, presenting with a moderately elevated level of LH/hCG bioactivity develop multiple neoplasms secondary to the endocrine abnormalities, including mammary gland tumors after the age of 9 months. The increased levels of circulating estradiol, progesterone, and prolactin of the TG females after puberty boost the lobuloalveolar development in the mammary gland resulting ultimately in the formation of estrogen and progesterone receptor-negative, malignant tumors. These tumors have a similar histopathology with those observed in TG mice with activated wnt/beta-catenin pathway, showing increased expression of beta-catenin, also a common finding in human breast tumors. Transdifferentiation is observed in mammary tumors of the hCGbeta TG mice, accompanied by abnormal expression of the Wnt genes in the tumorous and nontumorous mammary gland tissue. Specifically we found increased expression of Wnt5b in the TG mammary glands at the age of 3 months and up-regulation of Wnt7b and -5b in the subsequently appearing tumors. Importantly, hCG was found to up-regulate these wnt ligands in mouse mammary gland, independent of the changes in ovarian steroidogenesis. Thus, the hCGbeta-overexpressing TG mice represent a novel model that links enhanced hCG action to dysregulated wnt signaling in the mammary gland, resulting in beta-catenin-stabilizing mammary tumorigenesis. The novel finding of hCG up-regulating wnt7b and wnt5b could contribute to pregnancy-induced breast cancer in humans.

Translational Fusion of Two β-Subunits of Human Chorionic Gonadotropin Results in Production of a Novel Antagonist of the Hormone

The strategy of translationally fusing the alpha- and beta-subunits of human chorionic gonadotropin (hCG) into a single-chain molecule has been used to produce novel analogs of hCG. Previously we reported expression of a biologically active single-chain analog hCGalphabeta expressed using Pichia expression system. Using the same expression system, another analog, in which the alpha-subunit was replaced with the second beta-subunit, was expressed (hCGbetabeta) and purified. hCGbetabeta could bind to LH receptor with an affinity three times lower than that of hCG but failed to elicit any response. However, it could inhibit response to the hormone in vitro in a dose-dependent manner. Furthermore, it inhibited response to hCG in vivo indicating the antagonistic nature of the analog. However, it was unable to inhibit human FSH binding or response to human FSH, indicating the specificity of the effect. Characterization of hCGalphabeta and hCGbetabeta using immunological tools showed alterations in the conformation of some of the epitopes, whereas others were unaltered. Unlike hCG, hCGbetabeta interacts with two LH receptor molecules. These studies demonstrate that the presence of the second beta-subunit in the single-chain molecule generated a structure that can be recognized by the receptor. However, due to the absence of alpha-subunit, the molecule is unable to elicit response. The strategy of fusing two beta-subunits of glycoprotein hormones can be used to produce antagonists of these hormones.

Expression Of Human Chorionic Gonadotropin β-Subunit Type I Genes Predicts Adverse Outcome In Renal Cell Carcinoma

Expression of the free beta-subunit of human chorionic gonadotropin (hCGbeta) in malignant tumors is frequently associated with aggressive disease. The pretreatment serum concentration of hCGbeta is an independent prognostic variable in renal cell carcinoma (RCC). The three so-called type II genes (hCGbeta 3/9, 5, and 8) have been shown to be up-regulated in relation to type I genes (hCGbeta 6/7) in some malignant tumors. We developed a reverse transcription-polymerase chain reaction method for quantification of relative levels of the mRNAs for the two types of hCGbeta genes and studied the association between the expression in RCC tissue (n = 104) and clinical outcome. hCGbeta mRNA expression was detected in 40% (42 of 104) of the tumors, and in 40 of these (93%), this consisted of hCGbeta type I mRNA only, whereas type II hCGbeta mRNA was detected in two samples. hCGbeta mRNA expression was significantly associated with a shorter disease-specific (log-rank P = 0.023; median survival 1.4 versus 7.9 years) and overall survival (log-rank P = 0.011). In a Cox regression model, stage (P < 0.0001) and hCGbeta mRNA expression (P < 0.0001) were independent prognostic variables. We conclude that expression of type I hCGbeta genes indicates adverse prognosis in RCC.

Time-dependent folding of immunological epitopes of the human chorionic gonadotropin β-subunit

We have explored the possibility to use 14 different monoclonal antibodies in order to follow the formation of the respective epitopes during the biosynthesis of hCG subunits and their association in JEG-3 choriocarcinoma cells using pulse (30s to 5min)-chase (0–180min) experiments. We found central cystine knot epitope structures (epitope β1) to be formed immediately and simultaneously with epitopes on the protruding hCG-β loops 1 and 3. We found also differences in the time-dependent folding of β2 and β4 epitopes, which are highly overlapping structures on the loops 1+3. These differences were reinforced by decreasing the temperature during the pulse-chase experiments to 25°C. Moreover, we describe for the first time an intracellular intact hCG β-subunit form that showed the transient expression of the hCG-β-core fragment epitope β11 in the course of the maturation of this subunit which casts new light on the presence of hCG-β-core fragment in Down's syndrome, tumors and pregnancy.

False-Negative Pregnancy Test in Hydatidiform Mole

A 16-year-old girl (gravida 0, para 0) presented to the emergency department with a 2-week history of nausea, vomiting, vaginal spotting, and lower leg edema. On examination we found a palpable lower abdominal mass. The patient acknowledged recent sexual activity but denied having any sexually transmitted diseases. Molar pregnancy was suspected because of the typical “snowstorm” appearance observed with ultrasound examination. The quantitative serum β-subunit of human chorionic gonadotropin (β-hCG) concentration was 746.20 IU/L (reference interval, <0.5 to 2.90 IU/L; reportable interval, 0.5–1000 IU/L). The result of a qualitative urine hCG test, however, was negative. Human error was initially suspected as the cause of the negative result in the first qualitative urine hCG test. Two experienced and well-trained senior medical technologists repeated the urine hCG test twice, but the results were still negative. The patient’s nurse also indicated that she did not think there had been mislabeling of the urine sample. Because of this discrepancy, we …

Ten tandem repeats of β-hCG 109–118 enhance immunogenicity and anti-tumor effects of β-hCG C-terminal peptide carried by mycobacterial heat-shock protein HSP65

The β-subunit of human chorionic gonadotropin (β-hCG) is secreted by many kinds of tumors and it has been used as an ideal target antigen to develop vaccines against tumors. In view of the low immunogenicity of this self-peptide,we designed a method based on isocaudamer technique to repeat tandemly the 10-residue sequence X of β-hCG (109–118), then 10 tandemly repeated copies of the 10-residue sequence combined with β-hCG C-terminal 37 peptides were fused to mycobacterial heat-shock protein 65 to construct a fusion protein HSP65-X10-βhCGCTP37 as an immunogen. In this study, we examined the effect of the tandem repeats of this 10-residue sequence in eliciting an immune by comparing the immunogenicity and anti-tumor effects of the two immunogens, HSP65-X10-βhCGCTP37 and HSP65-βhCGCTP37 (without the 10 tandem repeats). Immunization of mice with the fusion protein HSP65-X10-βhCGCTP37 elicited much higher levels of specific anti-β-hCG antibodies and more effectively inhibited the growth of Lewis lung carcinoma (LLC) in vivo than with HSP65-βhCGCTP37, which should suggest that HSP65-X10-βhCGCTP37 may be an effective protein vaccine for the treatment of β-hCG-dependent tumors and multiple tandem repeats of a certain epitope are an efficient method to overcome the low immunogenicity of self-peptide antigens.

Time Profile of Appearance and Disappearance of Circulating Placenta-Derived mRNA in Maternal Plasma

Fetal RNA of placental origin has been detected in the plasma of pregnant women, but the timing of the first appearance and the detailed kinetics of postdelivery clearance of such circulating RNA have not been studied. To address the timing of the first appearance of circulating placental RNA, we collected serial maternal blood samples from 47 women who had conceived by assisted reproductive procedures. To address the postdelivery clearance kinetics, we collected serial postdelivery blood samples from 6 pregnant women who had delivered by cesarean section. Placenta-derived transcripts were sought by real-time quantitative reverse transcription-PCR. The earliest gestational age at which human placental lactogen and human chorionic gonadotropin beta-subunit mRNAs were detectable in a proportion of the pregnant women was the 4th week of gestation. The postdelivery study indicated that the median apparent half-life for the clearance of human placental lactogen mRNA was 14 min. Placenta-derived mRNA can be found in maternal plasma from very early on in gestation, suggesting a possible role for early noninvasive prenatal diagnosis or monitoring. The rapid kinetics of circulating placental mRNA suggest that its plasma concentrations may be used to monitor recent physiologic or pathologic events.

Preoperative Serum hCGβ as a Prognostic Marker in Primary Fallopian Tube Carcinoma

Objectives: It was the aim of this study to evaluate the prognostic value of the pretreatment serum concentrations of the β-subunit of human chorionic gonadotropin (hCGβ), CA 125 and tumour-associated trypsin inhibitor (TATI) in primary fallopian tube carcinoma (PFTC). Methods: The pretreatment serum concentrations of hCGβ, CA 125 and TATI were analyzed in serum samples from 60 women with a mean age of 61 years, treated for PFTC between 1985 and 2000. Of the 91 patients treated during this period, 31 were excluded because no serum sample was available. The patients were followed-up for recurrence and survival until February 14, 2003. The prognostic value of the serum markers were compared with those of stage, grade and histological type. Results: The median survival time was 27 months and the overall 5-year survival rate 33%. Stage and size of the residual tumour (<1 vs. ≧1 cm) predicted both overall and disease-free survival (p < 0.050). Histology (serous vs. others) (p = 0.023) also influenced overall survival. Overall 5-year survival was 38% when serum hCGβ was below 3.5 pmol/l, while it was 18% when the level was higher (p = 0.052). The corresponding disease-free 5-year survival was 38 and 20%, respectively (p = 0.014). Patients with CA 125 values above 1,017 kU/l had an overall 5-year survival of 39% as compared with 14% for those with lower values (p = 0.009), while the disease-free survival was 37 and 23%, respectively (p = 0.096). Serum TATI was not a prognostic marker. Serum concentrations of hCGβ and CA 125 correlated significantly with stage (p = 0.049 and p = 0.050, respectively). In multivariate Cox proportional hazards regression analysis, only hCGβ, stage and histology emerged as independent prognostic factors. Conclusions: Clearly elevated serum concentrations of hCGβ and CA 125 predict survival in fallopian tube carcinoma, but in multivariate analyses, only hCGβ is a prognostic factor independent of stage and histology.

Circulating Placental RNA in Maternal Plasma Is Associated with a Preponderance of 5′ mRNA Fragments: Implications for Noninvasive Prenatal Diagnosis and Monitoring

The molecular characteristics of placental RNA circulating in maternal plasma are unknown. We investigated the integrity of circulating placental RNA in maternal plasma and tested the relevance of plasma RNA integrity for noninvasive prenatal diagnosis. Six different placental transcripts and mRNA of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were quantified for the 5' and 3' regions in maternal plasma by 1-step real-time reverse transcription-PCR (RT-PCR) assays. This quantitative strategy was validated by 2-step RT-PCR and serial dilution experiments. The rates of detection by the 5' and 3' assays for the beta-subunit of human chorionic gonadotropin (beta hCG) were assessed in maternal plasma samples collected from different gestational periods. For 5 of the 7 genes, the plasma mRNA concentrations measured by the 5' amplicons were significantly higher than those measured by the corresponding 3' amplicons. Every transcript under study demonstrated a higher rate of detection in the 5' assay than in the 3' assay in maternal plasma. In particular, the detection rate of beta hCG mRNA in maternal plasma was increased throughout gestation when the 5' assay was used. Circulating placental RNA is associated with a preponderance of 5' mRNA fragments in maternal plasma. Apart from its intrinsic biological interest, this information could have important implications for the development of new assays targeting fetal RNA markers for noninvasive prenatal diagnosis and monitoring.

Cloning and sequencing of feline thyrotropin (fTSH): Heterodimeric and yoked constructs

The genes encoding the mature common glycoprotein α (CGA) and hormone-specific β subunits of feline thyroid stimulating hormone (fTSH) were cloned and sequenced. The feline CGA gene was cloned from RNA extracted from the feline pituitary gland by the reverse transcription polymerase chain reaction (RT-PCR). The gene fragment that encodes mature TSHβ was cloned from feline genomic DNA after direct polymerase chain reaction (PCR). In both cases, primers were based on the consensus sequences from TSH in other species. The resulting 510 bp PCR product for the CGA-subunit included the full coding sequence for the 96 amino acid mature subunit preceded by a 24 amino acid signal peptide. The 850 bp sequence of fTSHβ genomic DNA consisted of two coding exons, an intron of 418 bp, and a 60 bp signal sequence. The octapeptide immunoaffinity tag FLAG was added to 3′ end of the α gene to facilitate detection and purification. Both genes were cloned independently downstream from the EF1α promoter of the PEAK™ transfer vector to facilitate co-expression studies in PEAK™ cells (modified human embryonic kidney (HEK) cells). A single-chain analogue of fTSH termed yoked fTSH (yfTSH) was developed by fusing the nucleotides encoding the C-terminus of the β-subunit fused to the N-terminus of the α-subunit with DNA encoding the C-terminal peptide (CTP) of human chorionic gonadotropin β-subunit as a linker peptide. The resulting single-chain analogue encoded from N-terminus to C-terminus: β-CTP-α-FLAG. The resulting DNA sequence was cloned, sequenced, ligated and recloned into expression vector PEAK™. This report constitutes the first cloning and sequencing of the genes encoding the subunits of feline thyrotropin.