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Early pregnancy and multiparity are known to reduce the risk of women to develop breast cancer at menopause. Based on the knowledge that the differentiation of the breast induced by the hormones of pregnancy plays a major role in this protection, this work was performed with the purpose of identifying what differentiation-associated molecular changes persist in the breast until menopause. Core needle biopsies (CNB) obtained from the breast of 42 nulliparous (NP) and 71 parous (P) postmenopausal women were analyzed in morphology, immunocytochemistry and gene expression. Whereas in the NP breast, nuclei of epithelial cells were large and euchromatic, in the P breast they were small and hyperchromatic, showing strong methylation of histone 3 at lysine 9 and 27. Transcriptomic analysis performed using Affymetrix HG_U133 oligonucleotide arrays revealed that in CNB of the P breast, there were 267 upregulated probesets that comprised genes controlling chromatin organization, transcription regulation, splicing machinery, mRNA processing and noncoding elements including XIST. We concluded that the differentiation process induced by pregnancy is centered in chromatin remodeling and in the mRNA processing reactome, both of which emerge as important regulatory pathways. These are indicative of a safeguard step that maintains the fidelity of the transcription process, becoming the ultimate mechanism mediating the protection of the breast conferred by full-term pregnancy., (Copyright © 2011 UICC.)

The objective of this study was to comprehensively compare the genomic profiles in the breast of parous and nulliparous postmenopausal women to identify genes that permanently change their expression following pregnancy. The study was designed as a two-phase approach. In the discovery phase, we compared breast genomic profiles of 37 parous with 18 nulliparous postmenopausal women. In the validation phase, confirmation of the genomic patterns observed in the discovery phase was sought in an independent set of 30 parous and 22 nulliparous postmenopausal women. RNA was hybridized to Affymetrix HG_U133 Plus 2.0 oligonucleotide arrays containing probes to 54,675 transcripts, scanned and the images analyzed using Affymetrix GCOS software. Surrogate variable analysis, logistic regression, and significance analysis of microarrays were used to identify statistically significant differences in expression of genes. The false discovery rate (FDR) approach was used to control for multiple comparisons. We found that 208 genes (305 probe sets) were differentially expressed between parous and nulliparous women in both discovery and validation phases of the study at an FDR of 10% and with at least a 1.25-fold change. These genes are involved in regulation of transcription, centrosome organization, RNA splicing, cell-cycle control, adhesion, and differentiation. The results provide initial evidence that full-term pregnancy induces long-term genomic changes in the breast. The genomic signature of pregnancy could be used as an intermediate marker to assess potential chemopreventive interventions with hormones mimicking the effects of pregnancy for prevention of breast cancer., (©2011 AACR.)

Nematophagous fungi are soil-living fungi that are used as biological control agents of plant and animal parasitic nematodes. Their potential could be improved by genetic engineering, but the lack of information about the molecular background of the infection has precluded this development. In this paper we report that a subtilisin-like extracellular serine protease designated PII is an important pathogenicity factor in the common nematode-trapping fungus Arthrobotrys oligospora. The transcript of PII was not detected during the early stages of infection (adhesion and penetration), but high levels were expressed concurrent with the killing and colonization of the nematode. Disruption of the PII gene by homologous recombination had a limited effect on the pathogenicity of the fungus. However, mutants containing additional copies of the PII gene developed a higher number of infection structures and had an increased speed of capturing and killing nematodes compared to the wild type. The paralyzing activity of PII was verified by demonstrating that a heterologous-produced PII (in Aspergillus niger) had a nematotoxic activity when added to free-living nematodes. The toxic activity of PII was significantly higher than that of other commercially available serine proteases. This is the first report showing that genetic engineering can be used to improve the pathogenicity of a nematode-trapping fungus. In the future it should be possible to express recombinant subtilisins with nematicidal activity in other organisms that are present in the habitat of parasitic nematodes (e.g., host plant).

A new catalyst system for the enantioselective alpha-arylation of ketones is reported. This catalyst, prepared from Pd(2)(dba)(3) and a bulky dialkylphosphino-binaphthyl ligand, is able to effect the asymmetric arylation of ketone enolates with aryl bromides utilizing NaO(t)()Bu as base. These new catalysts enjoy much higher reactivity than previous systems; arylation reactions could be effected at room temperature with only 2 mol % of the Pd catalyst. The coupling of alpha-alkyl-alpha'-protected cyclopentanones proceeded in high yield, and the resulting quaternary stereogenic center was installed in up to 94% ee.

The nematode-trapping fungus Arthrobotrys oligospora was transformed to hygromycin resistance using the hygromycin-B phosphotransferase gene from Escherichia coli under the control of various heterologous fungal promoters. Plasmid DNA was introduced into fungal protoplasts by polyethylene glycol/CaCl2 treatment. Transformation frequencies varied between 1-6 transformants per microgram DNA. Seven out of 13 integration events analyzed from transformants were single copy integrations, whereas the remaining were multiple and more complex integrations. The addition of restriction enzymes during transformations increased the frequency of single copy integrations. Co-transformation, using the E. coli uidA gene encoding the beta-glucuronidase reporter gene under the control of an Aspergillus nidulans promoter, occurred at frequencies of up to 63%.

Two analogues of the antidiuretic drug [1-desamino,8-D-arginine]vasopressin (DDAVP), which have a glycosylated serine at position 4, have been prepared by Fmoc solid phase peptide synthesis. The glycosylated analogues had significantly higher bioavailabilities than the nonglycosylated [D-Tyr2,Ser4]DDAVP and DDAVP on intraintestinal administration in rat. The improved bioavailability resulted from an increased absorption from the small intestine and most likely from an increased stability toward enzymatic degradation, whereas plasma clearance was either unaffected or slightly increased by the glycosylation. The glycosylated analogues displayed only very low agonistic and antagonistic activities at the vasopressin V2-receptor. Conformational studies performed by 1H NMR spectroscopy did not reveal any major influence from glycosylation on the conformation of the peptide backbone. The lack of receptor binding displayed by the analogues is therefore most likely explained by steric repulsion between the carbohydrate moiety and the vasopressin receptor which prevents receptor binding.