Genetic polymorphims of estrogen receptor alpha −397 PvuII (T>C) and −351 XbaI (A>G) in a portuguese population: prevalence and relation with breast cancer susceptibility

Estrogen receptor alpha (ERα), that mediates the biologic effects of estrogen in estrogen-sensitive tissues like breast, is genetically polymorphic. To evaluate the association between ?397 PvuII (T>C) and ?351 XbaI (A>G) restriction fragment length polymorphisms (RFLPs) in intron 1 of ERα gene and susceptibility of breast cancer, we undertook a case–control study in BRCA1 185delAG and 5382insC/BRCA2 6174delT negative Portuguese women. The study population consisted of 107 patients with histological diagnosis of breast cancer and 121 women with no history of breast cancer. Genomic DNA was extracted from blood samples and genotyping analyses were performed by PCR–RFLP. XbaI polymorphism was associated with a significant reduced risk of breast cancer for carriers of the x allele in homozygozity (OR 0.178; 95 % CI 0.070–0.456; P < 0.001) or heterozigozity (OR 0.223; 95 % CI 0.089–0.561; P = 0.001). The PvuII polymorphism was associated with a non-significantly reduced risk. The combined analysis of PvuII and XbaI polymorphisms revealed none synergistic effect of the two genotypes, except for simultaneous carriers of pp and xx genotypes, that have a reduced risk of breast cancer (OR 0.226; 95 % CI 0.049–1.035; P = 0.044). The combination of PvuII and XbaI genotypes into haplotypes showed that carriers of two copies of the px (ppxx) haplotype had a reduced risk of breast cancer (OR 0.405; 95 % CI 0.194–0.843; P = 0.014), compared with PX (PPXX + PPXx + PpXX + PpXx) haplotypes. PvuII and XbaI polymorphisms were in linkage disequilibrium both in cases (D = 0.044, r² = 0.049, X² = 5.216, P = 0.022) and controls (D = 0.090, r² = 0.139, X² = 16.819, P < 0.001), but not in the entire sample population analyzed as a whole (D = 0.087, r² = 0.0076, X² = 1.733, P = 0.188). In conclusion, in this case–control study we found that ERα gene XbaI polymorphism may modify individual susceptibility for breast cancer in this population.     

Direct and continuous fluorescence-based measurements of Pyrococcus horikoshii DNA N-6 adenine methyltransferase activity

N-6 methylation of adenine destabilises duplex DNA and this can increase the proportion of DNA that dissociates into single strands. We have investigated utilising this property to measure the DNA adenine methyltransferase-catalyzed conversion of hemimethylated to fully methylated DNA through a simple, direct, fluorescence-based assay. The effects of methylation on the kinetics and thermodynamics of hybridisation were measured by comparing a fully methylated oligonucleotide product and a hemimethylated oligonucleotide substrate using a 13-bp duplex labeled on adjacent strands with a fluorophore (fluorescein) and quencher (dabcyl). Enzymatic methylation of the hemimethylated GATC site resulted in destabilisation of the duplex, increasing the proportion of dissociated DNA, and producing an observable increase in fluorescence. The assay provides a direct measurement of methylation rate in real time and is highly reproducible, with a coefficient of variance over 48 independent measurements of 3.6%. DNA methylation rates can be measured as low as 3.55 ± 1.84 fmol s⁻¹ in a 96-well plate format, and the assay has been used to kinetically characterise the Pyrococcus horikoshii DNA adenine methyltransferase.     

THE COMBINED USE OF WHOLE CUPHEA SEEDS CONTAINING MEDIUM CHAIN FATTY ACIDS AND AN EXOGENOUS LIPASE IN PIGLET NUTRITION

In search for an alternative for nutritional antimicrobials in piglet feeding, the effects of adding whole Cuphea seeds, as a natural source of medium chain fatty acids (MCFA), with known antimicrobial effects, and an exogenous lipase to a weaner diet were studied. The foregut flora, the gut morphology, some digestive parameters and the zootechnical performance of weaned piglets were investigated. Thirty newly weaned piglets, initial weight 7.0 ± 0.4 kg, were divided according to litter, sex and weight in two groups (control diet; Cuphea+lipase diet). The Cuphea seeds (lanceolata and ignea) (50 g kg^?1) were substituted for soybean oil (15 g kg^?1), Alphacell (25 g kg^?1) and soy protein isolate (10 g kg^?1) in the control diet. Also 500 mg kg^?1 microbial lipase was added to the Cuphea diet. The piglets were weighted individually on days 0, 3, 7, 14 and 16. Feed intake was recorded per pen during days 0 to 3, 3 to 7, 7 to 14 and 14 to 16. On day 7 five piglets of each experimental group were euthanized for counting the gastric and small intestinal gut flora and for gut morphology at two sites of the small intestine (proximal, distal). The results indicate a trend towards improved performances parameters by feeding Cuphea + lipase. The enzymic released MCFA (1.7 g kg^?1 fresh gastric contents) tended to decrease the number of Coliforms in the proximal small intestine, but increased the number in the stomach and distal small intestine. With Cuphea, the number of Streptococci was significantly lower in small intestine, but not in the stomach, while the number of Lactobacilli was significantly lower in the distal small intestine and tended to be lower in the stomach and proximal small intestine. No differences between the diets were noted for the total anaerobic microbial load in the stomach or in the gut. Feeding Cuphea+lipase resulted in a significantly greater villus height (distal small intestine) and a lesser crypt depth (proximal and distal small intestine) and greater villus/crypt ratio depth (proximal and distal small intestine). The intra-epithelial lymphocyte (IEL) counts per 100 enterocytes were significantly decreased in the proximal small intestine and tended to decrease in the distal small intestine by feeding the Cuphea+lipase diet. Both phenomena are indicative for a more healthy and better functional state of the mucosa. Present results are in line with foregoing research, showing that manipulation of the gut ecosystem by the enzymic in situ released MCFA in the stomach and foregut can result in improved performances of the piglets, which makes the concept a potential alternative for in-feed nutritional antibiotics.     

Characterization of a binding site for anionic phospholipids on KCNQ1

The KCNQ family of potassium channels underlie a repolarizing K(+) current in the heart and the M-current in neurones. The assembly of KCNQ1 with KCNE1 generates the delayed rectifier current I(Ks) in the heart. Characteristically these channels are regulated via G(q/11)-coupled receptors and the inhibition seen after phospholipase C activation is now thought to occur from membrane phosphatidylinositol (4,5)-bisphosphate (PIP(2)) depletion. It is not clear how KCNQ1 recognizes PIP(2) and specifically which residues in the channel complex are important. Using biochemical techniques we identify a cluster of basic residues namely, Lys-354, Lys-358, Arg-360, and Lys-362, in the proximal C terminus as being involved in binding anionic phospholipids. The mutation of specific residues in combination, to alanine leads to the loss of binding to phosphoinositides. Functionally, the introduction of these mutations into KCNQ1 leads to shifts in the voltage dependence of channel activation toward depolarized potentials and reductions in current density. Additionally, the biophysical effects of the charge neutralizing mutations, which disrupt phosphoinositide binding, mirror the effects we see on channel function when we deplete cellular PIP(2) levels through activation of a G(q/11)-coupled receptor. Conversely, the addition of diC8-PIP(2) to the wild-type channel, but not a PIP(2) binding-deficient mutant, acts to shift the voltage dependence of channel activation toward hyperpolarized potentials and increase current density. In conclusion, we use a combined biochemical and functional approach to identify a cluster of basic residues important for the binding and action of anionic phospholipids on the KCNQ1/KCNE1 complex.     

GIGANTEA regulates phytochrome A-mediated photomorphogenesis independently of its role in the circadian clock

GIGANTEA (GI) is a nuclear protein involved in the promotion of flowering by long days, in light input to the circadian clock, and in seedling photomorphogenesis under continuous red light but not far-red light (FR). Here, we report that in Arabidopsis (Arabidopsis thaliana) different alleles of gi have defects in the hypocotyl-growth and cotyledon-unfolding responses to hourly pulses of FR, a treatment perceived by phytochrome A (phyA). This phenotype is rescued by overexpression of GI. The very-low-fluence response of seed germination was also reduced in gi. Since the circadian clock modulates many light responses, we investigated whether these gi phenotypes were due to alterations in the circadian system or light signaling per se. In experiments where FR pulses were given to dark-incubated seeds or seedlings at different times of the day, gi showed reduced seed germination, cotyledon unfolding, and activity of a luciferase reporter fused to the promoter of a chlorophyll a/b-binding protein gene; however, rhythmic sensitivity was normal in these plants. We conclude that while GI does not affect the high-irradiance responses of phyA, it does affect phyA-mediated very-low-fluence responses via mechanisms that do not obviously involve its circadian functions.     

Determination of Protein-ligand Interactions Using Differential Scanning Fluorimetry

A wide range of methods are currently available for determining the dissociation constant between a protein and interacting small molecules. However, most of these require access to specialist equipment, and often require a degree of expertise to effectively establish reliable experiments and analyze data. Differential scanning fluorimetry (DSF) is being increasingly used as a robust method for initial screening of proteins for interacting small molecules, either for identifying physiological partners or for hit discovery. This technique has the advantage that it requires only a PCR machine suitable for quantitative PCR, and so suitable instrumentation is available in most institutions; an excellent range of protocols are already available; and there are strong precedents in the literature for multiple uses of the method. Past work has proposed several means of calculating dissociation constants from DSF data, but these are mathematically demanding. Here, we demonstrate a method for estimating dissociation constants from a moderate amount of DSF experimental data. These data can typically be collected and analyzed within a single day. We demonstrate how different models can be used to fit data collected from simple binding events, and where cooperative binding or independent binding sites are present. Finally, we present an example of data analysis in a case where standard models do not apply. These methods are illustrated with data collected on commercially available control proteins, and two proteins from our research program. Overall, our method provides a straightforward way for researchers to rapidly gain further insight into protein-ligand interactions using DSF.