Molecular analysis of the eighteen most frequent mutations in the BRCA1 gene in 63 Chilean breast cancer families

BRCA1 gene mutations account for nearly all families with multiple cases of both early onset breast and/or ovarian cancer and about 30% of hereditary breast cancer. Although to date more than 1,237 distinct mutations, polymorphisms, and variants have been described, several mutations have been found to be recurrent in this gene. We have analyzed 63 Chilean breast/ovarian cancer families for eighteen frequent BRCA1 mutations. The analysis of the five exons and two introns in which these mutations are located was made using mismatch PCR assay, ASO hybridization assay, restriction fragment analysis, allele specific PCR assay and direct sequentiation techniques. Two BRCA1 mutations (185delAG and C61G) and one variant of unknown significance (E1250K) were found in four of these families. Also, a new mutation (4185delCAAG) and one previously described polymorphism (E1038G) were found in two other families. The 185delAG was found in a 3.17% of the families and the others were present only in one of the families of this cohort. Therefore these mutations are not prominent in the Chilean population. The variant of unknown significance and the polymorphism detected could represent a founder effect of Spanish origin.     

Automated analysis of vapor diffusion crystallization drops with an X-ray beam

Crystallogenesis, usually based on the vapor diffusion method, is currently considered one of the most difficult steps in macromolecular X-ray crystallography. Due to the increasing number of crystallization assays performed by protein crystallographers, several automated analysis methods are under development. Most of these methods are based on microscope images and shape recognition. We propose an alternative method of identifying protein crystals: by directly exposing the crystallization drops to an X-ray beam. The resulting diffraction provides far more information than classical microscope images. Not only is the presence of diffracting crystals revealed, but also a first estimation of the space group, cell parameters, and mosaicity is obtained. In certain cases, it is also possible to collect enough data to verify the presence of a specific substrate or a heavy atom. All these steps are performed without the sometimes tedious necessity of removing crystals from their crystallization drop.     

A germin-like protein of wheat leaf apoplast inhibits serine proteases

A protein resistant to heat and proteolysis that inhibits serine proteases was isolated from wheat leaf apoplasts. Based on trypsin inhibition, its more active form was a 66-69 kDa oligomer. It was dissociated in an 18-21 kDa monomer having an amino terminal sequence identical to the Box A of germins and germin-like proteins. Like these proteins, it was glycosylated and showed manganese superoxide dismutase activity. The monomer displayed three forms when examined by 2D western blot: two of 19 kDa, pI 5.8 and 6.2; and one of 21 kDa, pI 5.8. It was found that the protein controls serine protease activity in the apoplast of plants challenged with the fungus Septoria tritici.     

Structural features of an arabinogalactan gum exudates from Spondias dulsis (Anacardiaceae)

The tree Spondias dulcis, located in Venezuela, exudes a light-brown gum. The polysaccharide, isolated from the original gum, contains galactose, arabinose, mannose, rhamnose, glucuronic acid, and its 4-O-methyl derivative. Application of chemical methods, in combination with 1D and 2D NMR spectroscopy afforded interesting structural features of the gum polysaccharide. The unequivocal presence of rhamnose in the polymer structure was confirmed by chemical and spectral data [1H (1.03 ppm); 13C (16.92 ppm)]. Also confirmed was the existence of 3-O- and 6-O-substitutes galactose residues by the spectral data correlations observed in Heteronuclear Multiple Quantum Coherence (HMQC) and Heteronuclear Multiple Bond Correlation (HMBC). Also observed were unequivocal resonances for beta-D-glucuronic acid and its 4-O-methyl derivative, and the presence of 3-O-alpha-L-arabinofuranose and 3-O-beta-L-arabinopyranose residues.     

Cytoskeleton of human mononuclear cells as a possible peripheral marker for phenylalanine neurotoxicity in PKU

In this work we tested human mononuclear cells as a peripheral marker to study neurotoxicity of phenylalanine (Phe). Slices of cerebral cortex of rats or human mononuclear cells were incubated with different concentrations of Phe and/or Ala in the presence of ³²P-orthophosphate, the cytoskeletal fraction was extracted, and the radioactivity incorporated into intermediate filament proteins was measured. Our results show that 2 mM Phe as well as 1 mM Ala are effective in increasing the ³²P in vitro incorporation into IFs in both tissues. When cerebral cortex slices or mononuclear cells were incubated with different concentrations of Phe and/or Ala, the effects on the ³²P in vitro incorporation into IF proteins was compatible with an antagonistic mechanism of action of the two amino acids on the enzymes of the phosphorylating system. In addition, these blood cells may be a possible peripheral marker to study neurotoxicity of Phe in patients with PKU.     

Evidence of Field-Evolved Resistance of Spodoptera frugiperda to Bt Corn Expressing Cry1F in Brazil That Is Still Sensitive to Modified Bt Toxins

Brazil ranked second only to the United States in hectares planted to genetically modified crops in 2013. Recently corn producers in the Cerrado region reported that the control of Spodoptera frugiperda with Bt corn expressing Cry1Fa has decreased, forcing them to use chemicals to reduce the damage caused by this insect pest. A colony of S. frugiperda was established from individuals collected in 2013 from Cry1Fa corn plants (SfBt) in Brazil and shown to have at least more than ten-fold higher resistance levels compared with a susceptible colony (Sflab). Laboratory assays on corn leaves showed that in contrast to SfLab population, the SfBt larvae were able to survive by feeding on Cry1Fa corn leaves. The SfBt population was maintained without selection for eight generations and shown to maintain high levels of resistance to Cry1Fa toxin. SfBt showed higher cross-resistance to Cry1Aa than to Cry1Ab or Cry1Ac toxins. As previously reported, Cry1A toxins competed the binding of Cry1Fa to brush border membrane vesicles (BBMV) from SfLab insects, explaining cross-resistance to Cry1A toxins. In contrast Cry2A toxins did not compete Cry1Fa binding to SfLab-BBMV and no cross-resistance to Cry2A was observed, although Cry2A toxins show low toxicity to S. frugiperda. Bioassays with Cry1AbMod and Cry1AcMod show that they are highly active against both the SfLab and the SfBt populations. The bioassay data reported here show that insects collected from Cry1Fa corn in the Cerrado region were resistant to Cry1Fa suggesting that resistance contributed to field failures of Cry1Fa corn to control S. frugiperda.     

Intravoxel Incoherent Motion Diffusion Weighted MR Imaging at 3.0 T: Assessment of Steatohepatitis and Fibrosis Compared with Liver Biopsy in Type 2 Diabetic Patients

ObjectiveTo evaluate the capability of intravoxel incoherent motion (IVIM) diffusion-weighted imaging (DWI) to assess steatohepatitis and fibrosis determined by histopathology in type 2 diabetic patients.MethodsFifty-nine type 2 diabetic patients (49 women, 10 men; mean age, 54 ± 9 years) were submitted to liver biopsy for the evaluation of non-alcoholic fatty liver disease (NAFLD) and underwent DWI on a 3.0T MR system using 10 b values. Institutional approval and patient consent were obtained. Pure molecular-based (D), perfusion-related (D*), and vascular fraction (f) were calculated using a double exponential model and least squares curve fitting. D, D*, and f were compared between patients with and without steatohepatitis and between patients with and without fibrosis. The variables were compared by using the Ranksum test and Student t-test.ResultsSteatohepatitis was observed in 22 patients and fibrosis in 16 patients. A lower D median (0.70 s/mm² vs. 0.83 s/mm², p<0.05) and a lower D* median (34.39 s/mm² vs. 45.23 s/mm², p<0.05) were observed among those with steatohepatitis. A lower D median (0.70 s/mm² vs. 0.82 s/mm², p<0.05) and a lower D* median (35.01 s/mm² vs. 44.76 s/mm², p=0.05) were also observed among those with fibrosis.ConclusionIVIM-DWI has the potential to aid in the characterization of steatohepatitis and fibrosis.