Familial breast/ovarian cancer and BRCA1/2 genetic screening: the role of immunohistochemistry as an additional method in the selection of patients.

Only 20-25% of families screened for BRCA1/2 mutations are found positive. Because only a positive result is informative, we studied the role of BRCA1/2 immunohistochemistry as an additional method for patient selection. From 53 high-risk-affected probands, 18 (34%) had available paraffin blocks of their tumors and were selected for this study. Mutation screening was done by conformation-sensitive gel electrophoresis and multiplex ligation-dependent probe amplification. For immunohistochemistry, 21 neoplastic specimens (15 breast carcinomas, 5 ovary neoplasms, and 1 rectal adenocarcinoma) were analyzed with BRCA1 (monoclonal antibody, Ab-1, oncogene) and BRCA2 (polyclonal antibody, Ab-2, oncogene) antibodies. Absence of the BRCA1 protein was confirmed in negative tumors by Western blotting. Seven patients were positive for BRCA1/2 mutations: 5 for BRCA1 and 2 for BRCA2. Four out of five positive patients had tumors negative for BRCA1 immunostaining, and the remaining 13 BRCA1-negative patients had positive BRCA1 immunostaining in all tumor samples. Sensitivity to predict for BRCA1 mutation carriers was 80%, and specificity was 100%, with a positive predictive value of 100% and a negative predictive value of 93%. This correlation was statistically significant (p=0.001). No correlation was observed for BRCA2. If larger studies confirm these results, high-risk patients with BRCA1-negative tumors should be screened first for this gene.     

Determination of the critical micelle concentration of surfactants using the fluorescent probe N-phenyl-1-naphthylamine

The fluorescence quantum yield of N-phenyl-1-naphthylamine (NPN) increases about 10-fold and the wavelength of maximum fluorescence emission is blue-shifted when this molecule partitions into the apolar core of micellar structures from the aqueous phase. This property allowed the utilization of NPN as a fluorescent indicator of micelle formation by 14 different surfactants belonging to the families of alkyltrimethylammonium halides, alkylsulfates, alkylbetaines, alkylglucosides, and bile salts. The critical micelle concentrations (CMCs) determined with NPN agreed well with literature values. In this work NPN was used at a concentration of 10(-6) M which allowed determination of CMCs in the range between approximately 10(-5) and greater than 10(-2) M. With high-sensitivity instrumentation considerably lower NPN concentrations can be used and consequently considerably lower CMCs can be rapidly and accurately determined.     

Bounding fluid viscosity and translational diffusion in a fluid lipid bilayer

Fluorescence recovery after photobleaching was used to investigate the translational diffusion of a fluorescent derivative of a membrane-spanning lipid in L phase multibilayers of 1-palmitoyl-2-oleoylphosphatidylcholine prepared in water and in glycerol. The translational diffusion coefficient in hydrated bilayers (D _w) ranged between 2 and 5x10^–8 cm²/s and in glycerinated bilayers (D _g) the range was between 3 and 24×10^–10 cm²/s between 10° and 40°C. These results are discussed in terms of models for diffusion in membranes.     

Effect of short-chain primary alcohols on fluidity and activity of sarcoplasmic reticulum membranes

Intramolecular excimer formation with the fluorescent probe 1,3-di(1-pyrenyl)propane, differential scanning calorimetry, and X-ray diffraction were used to assess the effect of ethanol, 1-butanol, and 1-hexanol on the bilayer organization in model membranes, sarcoplasmic reticulum (SR) lipids and native SR membranes. These alcohols have fluidizing effects on membranes and lower the main transition temperature of dimyristoylphosphatidylcholine (DMPC), but only 1-hexanol alters the cooperativity of the phase transition and significantly increases the thickness of DMPC bilayers. The interaction of the three alcohols with the SR Ca2+ pump was also investigated. Hydrolysis of ATP and coupled Ca2+ uptake are differently sensitive to the three alcohols. Whereas ethanol and 1-butanol inhibited the Ca2+ uptake, 1-hexanol stimulated it. Nevertheless, the energetic efficiency of the pump (Ca2+/ATP) is not significantly affected by ethanol or 1-hexanol, but uncoupling was observed with 1-butanol at high concentrations. The different effects of alcohols on the activity of SR membranes rule out an unitary mechanism of action on the basis of fluidity changes induced in the lipid bilayer. Depending on the chain length, the alcohols interact with the SR membranes in different domains, perturbing differently the Ca2+-pump activity.     

A comparison of the translational diffusion of a normal and a membrane-spanning lipid in Lα phase 1-palmitoyl-2-oleoylphosphatidylcholine bilayers

We have used the fluorescence recovery after photobleaching technique to study the translational diffusion, in L phase multibilayers of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), of fluorescent derivatives of 1-palmitoyl-2-oleoylphosphatidylethanolamine (NBD-POPE) and a membrane-spanning phosphatidylethanolamine (NBD-MSPE). The latter derivative was prepared from a membrane-spanning glycerol-dialkyl-glycerol tetraether lipid isolated from the thermophilic and acidophilic archaebacterium Sulfolobus solfataricus. The translational diffusion was examined between about 15° and 45°C. It is shown that over this temperature range the translational diffusion coefficient for NBD-MSPE is 2/3 that for NBD-POPE which spans only one monolayer of the bilayer. The result is interpreted in terms of existing models for translational diffusion in lipid membranes.Abbreviations D _t translational diffusion coefficient - FRAP fluorescence recovery after photobleaching - MSPE a membrane-spanning phosphatidylethanolamine derived from a glycerol-dialkyl-glycerol tetraether lipid isolated from Sulfolobus solfataricus - NBD 4-nitrobenz-2-oxa-1,3-diazolyl - PE phosphatidylethanolamine - POPC 1-palmitoyl-2-oleoylphosphatidylcholine - POPE 1-palmitoyl-2-oleoylphosphatidylethanolamine     

The thermodynamics of complex formation of cyclic tetra-aza-tetracetic acids

Enthalpy changes for the complexation of alkaline-earth and transition metals with three cyclic tetra-aza-tetracetic acids (cDOTA, cTRITA and cTETA) were obtained by continuous titration calorimetry. From these values and free energy data, the entropy changes for the same reactions were derived. The results show that these complexes are stabilised by both favourable enthalpy and entropy changes, except those of Mg²⁺ and those of Sr²⁺ and Ba²⁺ with cTETA. Generally, the entropy changes for the reactions of the alkaline-earth metals are higher than for the reactions of the non cyclic polyaminocarboxylic acids, but for the reactions of the transition metals the entropy changes are comparable for the cyclic and non cyclic ligands. These results are discussed in terms of a model of ‘cage’ coordination of the metals.The enthalpy changes decrease with the increase in size of the tetra-aza ring (except in the case of Cu²⁺) but no specific cavity size effect is noticeable. Consideration of the temperature-dependent and temperature-independent contributions to ΔH supports the idea that the number of coordinated nitrogen atoms and carboxylate groups vary along the series.     

Modulation of sarcoplasmic reticulum Ca2+-pump activity by membrane fluidity

Intramolecular excimerization of 1,3-di-1-pyrenylpropane [Py(3)Py] was used to assess the fluidity of sarcoplasmic reticulum membranes (SR); on the basis of the spectral data, the probe incorporates completely inside the membrane probably somewhere close to the polar head groups of phospholipid molecules, however not in the very hydrophobic core. The excimerization rate is very sensitive to lipid phase transitions, as revealed by thermal profiles of dimyristoyl-phosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC) bilayers. Cholesterol abolishes pretransitions and broadens the thermal profiles of the main transitions which vanish completely at 50 mol % sterol. Excimer formation in liposomes of SR total lipid extracts does not show any sharp transitions, as in the case of DMPC and DPPC. However, the plots display discontinuities at about 20 degrees C which are broadened by cholesterol and not observed at 50 mol % sterol. Also cholesterol has been incorporated in native SR membranes by an exchange technique allowing progressive enrichment without changing the phospholipid/protein molar ratio. As in liposomes, discontinuities of excimer formation at 20 degrees C are broadened by cholesterol enrichment. The full activity of uncoupled Ca2+-ATPase is only affected by cholesterol above a molar ratio to phospholipid of 0.4. However, a significant decrease in activity (about 20%) is only noticed at a ratio of 0.6 (the highest technically achieved); at this ratio, about 28 lipid molecules per Ca2+-ATPase are expected to be relatively free from cholesterol interaction. The vesicle structure is still intact at this high ratio, as judged from the absence of basal activity (not Ca2+ stimulated).(ABSTRACT TRUNCATED AT 250 WORDS)     

Transformation of passionfruit (Passiflora edulis fv flavicarpa Degener.) using Agrobacterium tumefaciens

Leaf and stem explants of passionfruit (Passiflora eadulis fv flavicarpa) were co-cultivated with a disarmed strain of Agrobacterium tunefaciens harbouring the co-integrate vector pMON200. Four plants of passionfruit were regenerated from leaf explants on agar-solidified Murashige and Skoog (1962) based medium containing 4.43 M 6-benzyl-aminopurine and supplemented with 86 M kanamycin sulphate. The four plants were rooted by transfer to MS based medium with 14.7 M 3-indolebutyric acid and 2.68 M -naphthyleneacetic acid for 7 d, followed by MS based medium lacking growth regulators. Both media used for rooting contained 172 M kanamycin sulphate. Rooted plants were potted and grown to maturity. Three of the plants synthesised nopaline and expressed neomycin phosphotransferase activity; DNA dot blot and polymerase chain reaction analyses confirmed the presence of the neomycin phosphotransferase gene in three plants.Abbreviations BAP 6-benzylaminopurine - CTAB hexadecy-Itrimethylammonium bromide - EDTA ethylenediaminetetraacetic acid - IBA 3-indolebutyric acid - MS Murashige and Skoog (1962) - NAA -naphthyleneacetic acid - NPTII neomycin phosphotransferase II - nptII neomycin phosphotransferase II gene - PCR polymerase chain reaction - SDS sodium dodecyl sulphate - Tris tris(hydroxymethyl)aminomethane