A rapid and concise setup for the fast screening of FRET pairs using bioorthogonalized fluorescent dyes

One of the most popular means to follow interactions between bio(macro)molecules is Forster resonance energy transfer (FRET). There is large interest in widening the selection of fluorescent FRET pairs especially in the region of the red/far red range, where minimal autofluorescence is encountered. A set of bioorthogonally applicable fluorescent dyes, synthesized recently in our lab, were paired (Cy3T/Cy5T; Cy1A/Cy3T and Cy1A/CBRD1A) based on their spectral characteristics in order to test their potential in FRET applications. For fast elaboration of the selected pairs we have created a bioorthogonalized platform based on complementary 17-mer DNA oligomers. The cyclooctynylated strands were modified nearly quantitatively with the fluorophores via bioorthogonal chemistry steps, using azide- (Cy1; CBRD1) or tetrazine-modified (Cy3; Cy5) dyes. Reactions were followed by capillary electrophoresis using a method specifically developed for this project. FRET efficiencies of the fluorescent dye pairs were compared both in close proximity (5' and 3' matched) and at larger distance (5' and 5' matched). The specificity of FRET signals was further elaborated by denaturation and competition studies. Cy1A/Cy3T and Cy1A/CBRD1A introduced here as novel FRET pairs are highly recommended for FRET applications based on the significant changes in fluorescence intensities of the donor and acceptor peaks. Application of one of the FRET pairs was demonstrated in live cells, transfected with labeled oligos. Furthermore, the concise installation of the dyes allows for efficient fluorescence modification of any selected DNA strands as was demonstrated in the construction of Cy3T labeled oligomers, which were used in the FISH-based detection of Helicobacter pylori

Activation of endogenous TRPV1 fails to induce overstimulation-based cytotoxicity in breast and prostate cancer cells but not in pain-sensing neurons

Vanilloids including capsaicin and resiniferatoxin are potent transient receptor potential vanilloid type 1 (TRPV1) agonists. TRPV1 overstimulation selectively ablates capsaicin-sensitive sensory neurons in animal models in vivo. The cytotoxic mechanisms are based on strong Na⁺ and Ca2 + influx via TRPV1 channels, which leads to mitochondrial Ca2 + accumulation and necrotic cell swelling. Increased TRPV1 expression levels are also observed in breast and prostate cancer and derived cell lines. Here, we examined whether potent agonist- induced overstimulation mediated by TRPV1 might represent a means for the eradication of prostate carcinoma (PC-3, Du 145, LNCaP) and breast cancer (MCF7, MDA-MB-231, BT-474) cells in vitro. While rat sensory neurons were highly vanilloid- sensitive, normal rat prostate epithelial cells were resistant in vivo. We found TRPV1 to be expressed in all cancer cell lines at mRNA and protein levels, yet protein expression levels were significantly lower compared to sensory neurons. Treatment of all human carcinoma cell lines with capsaicin didn't lead to overstimulation cytotoxicity in vitro. We assume that the low vanilloid-sensitivity of prostate and breast cancer cells is associated with low expression levels of TRPV1, since ectopic TRPV1 expression rendered them susceptible to the cytotoxic effect of vanilloids evidenced by plateau- type Ca2 + signals, mitochondrial Ca2 + accumulation and Na⁺- and Ca2 +-dependent membrane disorganization. Moreover, long- term monitoring revealed that merely the ectopic expression of TRPV1 stopped cell proliferation and often induced apoptotic processes via strong activation of caspase-3 activity. Our results indicate that specific targeting of TRPV1 function remains a putative strategy for cancer treatment

The Presence of a Novel Type of Surface Polysaccharide in \u3cem\u3eRhizobium meliloti\u3c/em\u3e Requires a New Fatty Acid Synthase-like Gene Cluster Involved in Symbiotic Nodule Development

Bacterial exopolysaccharide (EPS) and lipopolysaccharide (LPS) molecules have been shown to play important roles in plant‐bacterium interactions. Here we have demonstrated that the fix‐23 loci, which compensate for exo mutations during symbiotic nodule development, are involved in the production of a novel polysaccharide that is rich in 3‐deoxy‐D manno‐2‐octulosonic acid (Kdo) but is not the classical LPS. This molecule is likely to be a surface antigen since antiserum to whole Rhizobium meliloti cells reacts strongly with it, and since mutations in fix‐23 result in an inability to produce this polysaccharide and to bind bacteriophage 16‐3. It is likely that this Kdo‐rich polysaccharide is analogous to certain Escherichia coli K‐antigens which are anchored to the membrane via a phospholipid moiety. DNA sequence analysis of one gene cluster of this region revealed that the predicted protein products of six genes exhibit a high degree of homology and similar organization to those of the rat fatty acid synthase multifunctional enzyme domains

Towards the map based cloning of a recessive Fusarium resistance determinant from diploid Medicago sativa

A monogenic recessive Fusarium resistance determinant was identified in a diploid Medicago sativa population. The Fusarium susceptible and resistant phenotypes of the individuals in an F2 segregation population was inferred from a biological test using injured root test. The resulting phenotypes were used to position the Fusarium resistance determinant on linkage group 6 of the Medicago sativa genetic map. Using the DNA based molecular markers genome walk was initiated from both sides of the genetic region constructing two contigs which are about 0.5 centiMorgan (∼500 kilobasepairs) apart. The introduction of this genetic determinant into tetraploid alfalfa cultivars was unsuccessful since only triploid and pentaploid hybrids could be recovered from the 4×-2× crosses. The isolation of the gene and establishment of the function of this recessive Fusarium determinant is under progress

Pécs az egyháztörténet tükrében : Tanulmányok

A Pécsi Egyházmegye millenniuma tiszteletére, 2009 szeptemberében szervezett konferencia előadásai