In this article, a single-label separation-free fluorescence technique is presented as a potential screening method for cell-based receptor antagonists and agonists.The time-resolved fluorescence ...technique, quenching resonance energy transfer (QRET), relies on a single-labeled binding partner in combination with a soluble quencher. The quencher efficiently suppresses the luminescence of the unbound labeled ligand, whereas the luminescence of the bound fraction is not affected. This approach allows the development of cell-based screening assays in a simple and cost-effective manner. The authors have applied the technique to the screening of β2-adrenoreceptor (β2AR) antagonists and agonists in intact human embryonic kidney HEK293i cells overexpressing human β2-adrenergic receptors. Two antagonists (propranolol, alprenolol) and 2 agonists (metaproterenol, terbutaline) for β2AR were investigated in a displacement assay using europium(III)-labeled pindolol ligand. The assay Z′ values ranged from 0.68 to 0.78, the coefficient of variation was less than 10%, and the Ki values were 19 nM for propranolol and alprenolol and 14 and 5.9 μM for metaproterenol and terbutaline, respectively. The QRET technique with β2AR was also applied to LOPAC compound library screening, yielding nearly error-free recognition of known binders. This simple and cost-effective technique can be readily adapted to laboratory and industrial-scale screening.
Current treatment options for castration-resistant prostate cancer (CRPC) are limited. In this study, a high-throughput screen of 4910 drugs and drug-like molecules was performed to identify ...antiproliferative compounds in androgen ablated prostate cancer cells. The effect of compounds on cell viability was compared in androgen ablated LNCaP prostate cancer cells and in LNCaP cells grown in presence of androgens as well as in two non-malignant prostate epithelial cells (RWPE-1 and EP156T). Validation experiments of cancer specific anti-proliferative compounds indicated pinosylvin methyl ether (PSME) and tanshinone IIA as potent inhibitors of androgen ablated LNCaP cell proliferation. PSME is a stilbene compound with no previously described anti-neoplastic activity whereas tanshinone IIA is currently used in cardiovascular disorders and proposed as a cancer drug. To gain insights into growth inhibitory mechanisms in CRPC, genome-wide gene expression analysis was performed in PSME- and tanshinone IIA-exposed cells. Both compounds altered the expression of genes involved in cell cycle and steroid and cholesterol biosynthesis in androgen ablated LNCaP cells. Decrease in androgen signalling was confirmed by reduced expression of androgen receptor and prostate specific antigen in PSME- or tanshinone IIA-exposed cells. Taken together, this systematic screen identified a novel anti-proliferative agent, PSME, for CRPC. Moreover, our screen confirmed tanshinone IIA as well as several other compounds as potential prostate cancer growth inhibitors also in androgen ablated prostate cancer cells. These results provide valuable starting points for preclinical and clinical studies for CRPC treatment.
At the moment, there is no clear molecular explanation for the steeper decline in muscle performance after menopause or the mechanisms of counteractive treatments. The goal of this genome-wide study ...was to identify the genes and gene clusters through which power training (PT) comprising jumping activities or estrogen containing hormone replacement therapy (HRT) may affect skeletal muscle properties after menopause. We used musculus vastus lateralis samples from early stage postmenopausal (50-57 years old) women participating in a yearlong randomized double-blind placebo-controlled trial with PT and HRT interventions. Using microarray platform with over 24,000 probes, we identified 665 differentially expressed genes. The hierarchical clustering method was used to assort the genes. Additionally, enrichment analysis of gene ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways was carried out to clarify whether assorted gene clusters are enriched with particular functional categories. The analysis revealed transcriptional regulation of 49 GO/KEGG categories. PT upregulated transcription in “response to contraction”—category revealing novel candidate genes for contraction-related regulation of muscle function while HRT upregulated gene expression related to functionality of mitochondria. Moreover, several functional categories tightly related to muscle energy metabolism, development, and function were affected regardless of the treatment. Our results emphasize that during the early stages of the postmenopause, muscle properties are under transcriptional modulation, which both PT and HRT partially counteract leading to preservation of muscle power and potentially reducing the risk for aging-related muscle weakness. More specifically, PT and HRT may function through improving energy metabolism, response to contraction as well as by preserving functionality of the mitochondria.
The DNA sequencing of the entire nuclear genomes from Arabidopsis, rice and poplar has facilitated the broad-adoption of contemporary research techniques that extend far beyond the study of ...individual genes. New post-genomic technologies such as microarray based genome-scale gene expression profiling and proteome analysis are absolutely dependent on deep sequence coverage of the gene-space, but have an immense potential to drive the research community in exciting new directions. Facets of many crop genomes currently preclude them from complete sequencing, but the broadest adoption of post-genomic technologies is essential to make in-roads in plant biotechnology and crop improvement. A variety of technologies are available that can be used to establish a genomics foothold in even the most recalcitrant of crop plant species. In this review we address the fundamental technologies that are being widely adopted within the crop-plant research community to gain such a foothold. By considering primarily the expressed sequence tag resources, we have explored how even moderately simple genomic resources may be exploited in molecular marker development, candidate gene selection and microarray-based gene expression profiling.
Excellent long-term results are observed in patients with clinical stage I high-risk nonseminomatous germ-cell tumors after one cycle of adjuvant modified-BEP chemotherapy.
To report the long-term ...results of adjuvant treatment with one cycle of modified bleomycin, etoposide, and cisplatin (BEP) in patients with clinical stage I (CS I) nonseminomatous germ-cell tumors (NSGCT) at high risk of relapse.
In a single-arm, phase II clinical trial, 40 patients with CS I NSGCT with vascular invasion and/or >50% embryonal cell carcinoma in the orchiectomy specimen received one cycle of adjuvant BEP (20 mg/m2 bleomycin as a continuous infusion over 24 h, 120 mg/m2 etoposide and 40 mg/m2 cisplatin each on days 1–3). Primary end point was the relapse rate.
Median follow-up was 186 months. One patient (2.5%) had a pulmonary relapse 13 months after one BEP and died after three additional cycles of BEP chemotherapy. Three patients (7.5%) presented with a contralateral metachronous testicular tumor, and three (7.5%) developed a secondary malignancy. Three patients (7.5%) reported intermittent tinnitus and one had grade 2 peripheral polyneuropathy (2.5%).
Adjuvant chemotherapy with one cycle of modified-BEP is a feasible and safe treatment of patients with CS I NSGCT at high risk of relapse. In these patients, it appears to be an alternative to two cycles of BEP and to have a lower relapse rate than retroperitoneal lymph node dissection. If confirmed by other centers, 1 cycle of adjuvant BEP chemotherapy should become a first-line treatment option for this group of patients.
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