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Triptorelin, a synthetic gonadotrophin-releasing hormone (GnRH), is mainly used in the clinical treatment of prostate cancer. The mechanism initially stimulates luteinizing hormone (LH) and testosterone secretion followed by suppression, resulting in a reduction in cancer progression. However, GnRHs are prohibited in doping control because of the indirect surge of LH and testosterone. Therefore, GnRH analog detection and confirmation are enforced by World Anti-Doping Agency (WADA) requirements. The effects of triptorelin on LH and endogenous steroid levels in urine and serum of five prostate cancer patients taking triptorelin for the first time were investigated and compared with leuprorelin. The samples were collected at 0.0 h, 3.0 h, 6.0 h, 1 month, and 3 months later after drug administration. The effect of triptorelin on LH levels was measured using a sandwich enzyme-linked immunoassay (ELISA). Testosterone and endogenous steroid levels were monitored using gas chromatography coupled with mass spectrometry (GC/MS). Triptorelin showed an advantage over leuprorelin on LH and testosterone suppression, which is preferable to use for prostate cancer treatment. In this study, triptorelin (5-10), a unique in vivo metabolite, was found in urine and serum and verified with synthetic triptorelin (5-10). The metabolite was analyzed using liquid chromatography combined with Orbitrap (LC-Orbitrap) and liquid chromatography coupled with ion trap/time-of-flight mass spectrometry (LC/MS-IT-TOF). When triptorelin levels are undetectable, the presence of triptorelin (5-10) in human urine can be used as evidence that triptorelin is being misused in doping control., (© 2025 The Author(s). Drug Testing and Analysis published by John Wiley & Sons Ltd.)

Triptorelin and leuprorelin are synthetic gonadotrophin-releasing hormones (GnRH) that are on the World Anti-Doping Agency (WADA) list of prohibited substances. To investigate the possible in vivo metabolites of triptorelin and leuprorelin in humans compared to previously reported in vitro metabolites, excreted urine from five patients treated with either triptorelin or leuprorelin was analyzed by liquid chromatography coupled with ion trap/time-of-flight mass spectrometry (LC/MS-IT-TOF). The addition of dimethyl sulfoxide (DMSO) to the mobile phase was found to enhance the detection sensitivity of certain GnRH analogs. The method was validated, and the limit of detection (LOD) was found at 0.02-0.08 ng/mL. Using this method, a novel new metabolite of triptorelin was discovered in the urine of all subjects up to 1 month after triptorelin administration, but it was not observed in the urine of subjects before drug administration. The limit of detection was estimated to be 0.05 ng/mL. The structure of the metabolite, triptorelin (5-10), is proposed from bottom-up mass spectrometry analysis. The discovery of in vivo triptorelin (5-10) can possibly be used as supporting evidence of triptorelin misuse in athletes.

Chondrogenic differentiation of embryonic stem cells (ESCs) via embryoid bodies (EBs) is an established model to investigate chondrogenesis signaling pathways and molecular mechanisms in vitro. Our aim has been to improve upon the number of differentiated cells needed for the in vitro development of functional cartilage. Chondrogenic differentiation of buffalo ESCs was modulated by bone morphogenetic protein 2 (BMP-2), fibroblast growth factor 10 (FGF-10), transforming growth factor-beta1 (TGF-β1 ) individually and their combination. ESCs differentiation into chondrocytes was characterized by the appearance of Alcian blue-stained nodules and the expression of cartilage-associated genes (RT-PCR) and protein (immunocytochemistry). BMP-2 or FGF-10 treatment enhanced chondrogenic differentiation, whereas TGF-β1 treatment inhibited buffalo ESC-derived chondrogenesis. The combination of BMP-2 and FGF-10 was the most effective treatment. This treatment resulted in a higher number of Alcian blue-positive nodules by 15.2-fold, expression of the mesenchymal cell marker scleraxis gene by 3.25-fold, and the cartilage matrix protein collagen II gene and protein 1.9- and 7-fold, respectively, compared to the untreated control group. Chondrogenesis was also recapitulated from mesenchymal and chondrogenic progenitor cells, resulting in the establishment of mature chondrocytes. Thus, buffalo ESCs can be successfully triggered in vitro to differentiate into chondrocyte-like cells by specific growth factors, which may provide a novel in vitro model for further investigation of the regulatory mechanism(s) involved., (© 2013 International Federation for Cell Biology.)

Embryonic stem (ES) cells derived from mammalian embryos have the ability to form any terminally differentiated cell of the body. We herein describe production of parthenogenetic buffalo (Bubalus Bubalis) blastocysts and subsequent isolation of an ES cell line. Established parthenogenetic ES (PGES) cells exhibited diploid karyotype and high telomerase activity. PGES cells showed remarkable long-term proliferative capacity providing the possibility for unlimited expansion in culture. Furthermore, these cells expressed key ES cell-specific markers defined for primate species including stage-specific embryonic antigen-4 (SSEA-4), tumor rejection antigen-1-81 (TRA-1-81), and octamer-binding transcription factor 4 (Oct-4). In vitro, in the absence of a feeder layer, cells readily formed embryoid bodies (EBs). When cultured for an extended period of time, EBs spontaneously differentiated into derivatives of three embryonic germ layers as detected by PCR for ectodermal (nestin, oligodendrocytes, and tubulin), mesodermal (scleraxis, alpha-skeletal actin, collagen II, and osteocalcin) and endodermal markers (insulin and alpha-fetoprotein). Differentiation of PGES cells toward chondrocyte lineage was directed by supplementing serum-containing media with ascorbic acid, beta-glycerophosphate, and dexamethasone. Moreover, when PGES cells were injected into nude mice, teratomas with derivatives representing all three embryonic germ layers were produced. Our results suggest that the cell line isolated from a parthenogenetic blastocyst holds properties of ES cells, and can be used as an in vitro model to study the effects of imprinting on cell differentiation and as an a invaluable material for extensive molecular studies on imprinted genes., ((c) 2007 Wiley-Liss, Inc.)

We have previously shown that the flowers of neem tree (Azadirachta indica A. Juss, family Meliaceae), Thai variety, strongly induced the activity of glutathione S-transferase (GST) while resulting in a significant reduction in the activities of some cytochrome P(450)-dependent monooxygenases in rat liver, and possess cancer chemopreventive potential against chemically-induced mammary gland and liver carcinogenesis in rats. In the present study, 2 chemicals possessing strong QR inducing activity were fractionated from neem flowers using a bioassay based on the induction of QR activity in mouse hepatoma Hepa 1c1c7 cultured cells. Spectroscopic characteristics revealed that these compounds were nimbolide and chlorophylls, having CD (concentration required to double QR specific activity) values of 0.16 and 3.8 mug/ml, respectively. Nimbolide is a known constituent of neem leaves, but was found for the first time here in the flowers. Both nimbolide and chlorophylls strongly enhanced the level of QR mRNA in Hepa 1c1c7 cells, as monitored by northern blot hybridization, indicating that the mechanism by which these constituents of neem flowers induced QR activity is the induction of QR gene expression. These findings may have implication on cancer chemopreventive potential of neem flowers in experimental rats previously reported.

The tegument of bile-dwelling Fasciola gigantica is the interfacing layer that helps the parasite to maintain its homeostasis, and evade the hostile environment, including the host's immune attacks. The tegument is a syncytial layer about 10 mm thick, that is formed by the fusion of cytoplasmic processes of tegument cells, whose soma lie underneath the two muscle layers. The surface of the tegument is highly folded and invaginated into numerous ridges, pits and spines, which help to increase the surface area of the tegument for the absorption and exchanging of molecules, as well as for attachment. The outer membrane covering the tegument is a trilaminate sheet about 12 nm thick, and coated with a carbohydrate-rich glycocalyx layer that also bears high negative charges. Some host molecules may also be adsorbed onto this layer. These unique characteristics enable the parasite to evade the antibody-dependent cell-mediated cytotoxicity (ADCC) reaction exerted by the host. The outer membrane and glycocalyx is continuously replaced by the reserved membrane synthesized and stored in secretory granules of tegument cells, that are transported via cell processes towards the tegument by microtubules. The basal membrane of the tegument is trilaminate and invaginated to form membrane infoldings with closely aligned mitochondria. The tegument cytoskeleton is composed of a highly cross-linked network of 4-6 nm knobby microtrabecular fibers, bundles of intermediate filaments, microtubules that splay out from the tegument cells' processes. Major proteins of the cytoskeleton are actin, paramyosin and tubulin. The flukes' antigens that can elicit strong immunological responses in animal hosts are synthesized and released mainly from the tegument and the cecum. The majority of antigens derived from the surface membrane and the tegument are of MW 97, 66, 58, 54, 47 and 14 kDa, while those released from the cecum are cysteine proteases of MW 27, 26 kDa. Monoclonal antibodies have been raised against some of these antigens, and have been employed in immunodiagnosis of the infection. From the protection conferred to animal models and the in vitro killing assays of young parasites by specific antibodies, candidate vaccines could be selected from these antigens, such as, an antioxidant enzyme, glutathione-S-transferase, the digestive enzyme cysteine proteases, the surface-tegument proteins, such as fatty acid binding protein (14 kDa), membrane proteins (at 66 kDa), as well as muscle protein paramyosin, and hemoprotein. Ongoing research have been directed at deciphering the genetic codes and the syntheses of some of these antigens by recombinant DNA technology.

A monoclonal antibody (MoAb) 1C12 that reacts with a 66 kDa surface tegumental (ST) antigen of adult worms of Fasciola gigantica was used to detect circulating antigen in sera of experimentally and naturally infected cattle. A combination of rabbit anti ST-antigens and MoAb 1C12 were used to capture and detect the circulating antigen in sandwich ELISA. The dilutions of 1:1,000 of rabbit anti ST-antigens and 1:100 for MoAb 1C12 were used to reduce cross-reactivity with other trematodes' antigens. The circulating antigen of F. gigantica was demonstrated in sera of all experimentally infected animals as early as the first week after the infection, and it remained detectable until the experiment was terminated at week 32 after the infection. Of the 97 serum samples from naturally infected cattle, the sensitivity of 86.6% was observed when the cut-off point was calculated from 32 serum specimens from uninfected control calves. The sensitivity increased to 100% when the commercial fetal calf and trematode-free baby calves sera were used for calculation of the control cut-off point. Based on these results, the combination of rabbit anti ST-antigens and MoAb 1C12 sandwich ELISA appeared to be sensitive, specific, and applicable in the immunodiagnosis of fasciolosis in cattle for epidemiological study and monitoring of chemotherapeutic efficacy.

Two monoclonal antibodies (McAbs) were produced from BALB/c mice hyperimmunized with tegumental extract of Schistosoma japonicum (Chinese strain). The two McAbs were characterized with regard to antibody isotype, antigen binding specificity and parasite stage specificity. One McAb, 8G9-5, was identified as IgM, whereas the other McAb, 9E7, was determined to be IgG2a. Immunoblotting assay indicated that McAb 8G9-5 binds strongly to the band of tegumental antigens of Mr 64 kDa but also binds weakly to other bands at 116, 105, 97, 54, 50, 47 and 45 kDa, whereas 9E7 McAb reacts specifically at Mr 54 kDa. Anatomical localization of the antigens in the adult worm by indirect immunofluorescence assay indicated that the target epitopes of McAb 8G9-5 are in the intra-tegumental structure, whereas the McAb 9E7 epitope is on the surface membrane. The two McAbs also react at similar sites within the teguments of schistosomula and lung worms.

Human placentae obtained early in pregnancy or at full term were examined for RNA content per cell, RNA polymerase types and activities, and chromatin template availability. The RNA:DNA ratio fell from 0.7 at 15 to 20 weeks to 0.4 at 40 weeks of pregnancy. Since RNase activities were similar at both times, the reduction in RNA content was attributed not to increased degradation, but to reduced synthesis. At both stages of pregnancy, about 55 to 60 per cent of the RNA polymerase activity in isolated placental nuclei was accounted for by RNA polymerase II, as judged by suppression of activity with alpha-amanitin and by separation of the extracted polymerases on DEAE-Sephadex. The relative roles of changes in polymerase activity and template availability were measured in nuclei from 20- and 40-week placentae. Nuclei showed 20 per cent greater polymerase activity in full-term than in early placentae, but the template availability of isolated chromatin for transcription by RNA polymerase II was 70 per cent less at full term. We conclude that the reduced amount of RNA per cell in the full-term placenta is due to reduced template availability that more than offsets the slight increase in polymerase activity.