Reduced expression of NGEP is associated with high-grade prostate cancers: a tissue microarray analysis

New gene expressed in prostate (NGEP) is a newly diagnosed prostate-specific gene that is expressed only in normal prostate and prostate cancer cells. Discovery of tissue-specific markers may promote the development of novel targets for immunotherapy of prostate cancer. In the present study, the staining pattern and clinical significance of NGEP were evaluated in a series of prostate tissues composed of 123 prostate cancer, 19 high-grade prostatic intraepithelial neoplasia and 44 samples of benign prostate tissue included in tissue microarrays using immunohistochemistry. Our study demonstrated that NGEP localized mainly in the apical and lateral membranes and was also partially distributed in the cytoplasm of epithelial cells of normal prostate tissue. All of the examined prostate tissues expressed NGEP with a variety of intensities; the level of expression was significantly more in the benign prostate tissues compared to malignant prostate samples (P value <0.001). Among prostate adenocarcinoma samples, a significant and inverse correlation was observed between the intensity of NGEP expression and increased Gleason score (P = 0.007). Taken together, we found that NGEP protein is widely expressed in low-grade to high-grade prostate adenocarcinomas as well as benign prostate tissues, and the intensity of expression is inversely proportional to the level of malignancy. NGEP could be an attractive target for immune-based therapy of prostate cancer patients as an alternative to the conventional therapies particularly in indolent patients.     

The Arabidopsis ESCRT protein�Cprotein interaction network

In yeast, endosomal sorting of monoubiquitylated transmembrane proteins is performed by a subset of the 19 "class E vacuolar protein sorting" proteins. The core machinery consists of 11 proteins that are organised in three complexes termed ESCRT I-III (endosomal sorting complex required for transport I-III) and is conserved in eukaryotic cells. While the pathway is well understood in yeast and animals, the plant ESCRT system is largely unexplored. At least one sequence homolog for each ESCRT component can be found in the Arabidopsis genome. Generally, sequence conservation between yeast/animals and the Arabidopsis proteins is low. To understand details about participating proteins and complex organization we have performed a systematic pairwise yeast two hybrid analysis of all Arabidopsis proteins showing homology to the ESCRT core machinery. Positive interactions were validated using bimolecular fluorescence complementation. In our experiments, most putative ESCRT components exhibited interactions with other ESCRT components that could be shown to occur on endosomes suggesting that despite their low homology to their yeast and animal counterparts they represent functional components of the plant ESCRT pathway.     

Targeting host cell furin proprotein convertases as a therapeutic strategy against bacterial toxins and viral pathogens

Pathogens or their toxins, including influenza virus, Pseudomonas, and anthrax toxins, require processing by host proprotein convertases (PCs) to enter host cells and to cause disease. Conversely, inhibiting PCs is likely to protect host cells from multiple furin-dependent, but otherwise unrelated, pathogens. To determine if this concept is correct, we designed specific nanomolar inhibitors of PCs modeled from the extended cleavage motif TPQRERRRKKR downward arrowGL of the avian influenza H5N1 hemagglutinin. We then confirmed the efficacy of the inhibitory peptides in vitro against the fluorescent peptide, anthrax protective antigen (PA83), and influenza hemagglutinin substrates and also in mice in vivo against two unrelated toxins, anthrax and Pseudomonas exotoxin. Peptides with Phe/Tyr at P1' were more selective for furin. Peptides with P1' Thr were potent against multiple PCs. Our strategy of basing the peptide sequence on a furin cleavage motif known for an avian flu virus shows the power of starting inhibitor design with a known substrate. Our results confirm that inhibiting furin-like PCs protects the host from the distinct furin-dependent infections and lay a foundation for novel, host cell-focused therapies against acute diseases.     

Chaga mushroom extract inhibits oxidative DNA damage in lymphocytes of patients with inflammatory bowel disease

Inflammatory Bowel Disease (IBD) is partly caused by oxidative stress from free radicals and reduced antioxidant levels. Using hydrogen peroxide to induce oxidative stress in vitro in peripheral lymphocytes we investigated the induction of DNA damage supplemented with ethanolic extract of Chaga mushroom as a protective antioxidant. Lymphocytes were obtained from 20 IBD patients and 20 healthy volunteers. For treatment, a constant H_{2}O_{2 } dose (50 microg/ml) was used with variable doses of Chaga extract (10-500 microg/ml). DNA damage was evaluated in 50 cells per individual and dose using the Comet assay (making 1000 observations per experimental point ensuring appropriate statistical power). Chaga supplementation resulted in a 54.9% (p < 0.001) reduction of H_{2}O_{2 } induced DNA damage within the patient group and 34.9% (p < 0.001) within the control group. Lymphocytes from Crohn's disease (CD) patients had a greater basic DNA damage than Ulcerative Colitis (UC) patients (p < 0.001). Conclusively, Chaga extract reduces oxidative stress in lymphocytes from IBD patients and also healthy individuals when challenged in vitro. Thus, Chaga extract could be a possible and valuable supplement to inhibit oxidative stress in general.     

Hybrid molecules containing benzo[4,5]imidazo[1,2-d][1,2,4]thiadiazole and alpha-bromoacryloyl moieties as potent apoptosis inducers on human myeloid leukaemia cells

The synthesis and biological activity of a series of hybrids 1-5 prepared combining a benzo[4,5]imidazo[1,2-d][1,2,4]thiadiazole and different benzoheterocyclic alpha-bromoacryloyl amides have been described and their structure-activity relationships discussed. All these hetero-bifunctional compounds were highly cytotoxic against the human myeloid leukaemia cell lines HL-60 and U937 (IC(50) 0.24-1.72microM), significantly superior to that of both alkylating units alone. In human myeloid leukaemia HL-60 cells we observed that these compounds suppress survival and proliferation by triggering morphological changes and internucleosomal DNA fragmentation characteristic of apoptotic cell death. The apoptosis induced by these compounds is mediated by caspase-3 activation and is also associated to an early release of cytochrome c from the mitochondria.     

Hybrid molecules between distamycin A and active moieties of antitumor agents

The DNA minor groove is an attractive target for the design and development of molecules able to specifically recognize predetermined DNA sequences. The pyrrole-amide skeleton of distamycin A has been also used as DNA sequence selective vehicle for the delivery of alkylating functions to DNA targets. Selectivity for specific sequences may be of particular importance in affecting the activity of regulatory genes (oncogenes and tumor suppressor genes). Recent work on a number of hybrid compounds, in which known antitumor compounds or simple active moieties of known antitumor agents have been tethered to distamycin frame or hairpin polyamides derived from distamycin, is reviewed. The DNA alkylating and growth inhibition activities against several tumor cell lines are reported and discussed in terms of their structural differences in relation to both the number of N-methyl pyrrolic rings and the type of the alkylating unit tethered to the oligopyrrolic frame.     

Identifying nonselective hits from a homogeneous calcium assay screen

The authors used a homogeneous calcium dye kit with a cell line transfected using a recombinant protein construct to screen a 50,000 compound library for G-protein coupled receptor (GPCR) agonists. Only 1 of the 365 primary hits activated Gq-coupled GPCRs, as shown using IP-ONE HTRF. Furthermore, an agonist screen against the entire compound library and same heterologous cell line using AequoScreen technology generated no false positives and identified the same positive hit. Next, a multiplex assay composed of both Fluo-3 and Fura-2-loaded cells identified 1 false positive and the same true-positive hit out of the 365 primary hits. Finally, rescreening the 365 primary hits against the parental cell line loaded using the homogeneous calcium dye kit confirmed the specificity of the same true-positive hit only. In summary, the results suggest that AequoScreen technology, IP-ONE HTRF, and multiplex assays are unique, orthogonal technologies to identify nonspecific hits.