Ontogeny of human Ia antigens

Indirect immunofluorescence (IIP) staining of tissues from human fetuses (ages ranging from 8 to 32 weeks of intrauterine life) with monoclonal antibodies (MoAb) to monomorphic determinants of Ia antigens and HLA-A,B,C antigens has shown that both types of antigens are already detectable in tissues of 8-week-old fetuses. Ia antigens and HLA-A,B,C antigens reach their almost-complete tissue distribution after 32 and 24 weeks of intrauterine life, respectively. The structure of Ia antigens synthesized by fetal thymus cells is similar to that of B-lymphoid cell-derived Ia antigens. Ia antigen-bearing thymic fetal cells can stimulate allogeneic lymphocytes in mixed lymphocyte reactions (MLRs). These reactions are blocked by monoclonal antibodies to monomorphic determinants of human Ia antigens and of HLA-A,B, antigens.     

Charge state estimation for tandem mass spectrometry proteomics

High-throughput protein analysis by tandem mass spectrometry produces anywhere from thousands to millions of spectra that are being used for peptide and protein identifications. Though each spectrum corresponds only to one charged peptide (ion) state, repetitive database searches of multiple charge states are typically conducted since the resolution of many common mass spectrometers is not sufficient to determine the charge state. The resulting database searches are both error-prone and time-consuming. We describe a straightforward, accurate approach on charge state estimation (CHASTE). CHASTE relies on fragment ion peak distributions, and by using reliable logistic regression models, combines different measurements to improve its accuracy. CHASTE's performance has been validated on data sets, comprised of known peptide dissociation spectra, obtained by replicate analyses of our earlier developed protein standard mixture using ion trap mass spectrometers at different laboratories. CHASTE was able to reduce number of needed database searches by at least 60% and the number of redundant searches by at least 90% virtually without any informational loss. This greatly alleviates one of the major bottlenecks in high throughput peptide and protein identifications. Thresholds and parameter estimates can be tailored to specific analysis situations, pipelines, and instrumentations. CHASTE was implemented in Java GUI-based and command-line-based interfaces.     

Endothelin-1-induced prostaglandin E2-EP2, EP4 signaling regulates vascular endothelial growth factor production and ovarian carcinoma cell invasion

Cyclooxygenase (COX)-1- and COX-2-derived prostaglandins are implicated in the development and progression of several malignancies. We have recently demonstrated that treatment of ovarian carcinoma cells with endothelin-1 (ET-1) induces expression of both COX-1 and COX-2, which contributes to vascular endothelial growth factor (VEGF) production. In this study, we show that in HEY and OVCA 433 ovarian carcinoma cells, ET-1, through the binding with ETA receptor (ETAR), induces prostaglandin E2 (PGE2) production, as the more represented PG types, and increases the expression of PGE2 receptor type 2 (EP2) and type 4 (EP4). The use of pharmacological EP agonists and antagonists indicates that ET-1 and PGE2 stimulate VEGF production principally through EP2 and EP4 receptors. At the mechanistic level, we prove that the induction of PGE2 and VEGF by ET-1 involves Src-mediated epidermal growth factor receptor transactivation. Finally, we demonstrate that ETAR-mediated activation of PGE2-dependent signaling participates in the regulation of the invasive behavior of ovarian carcinoma cells by activating tumor-associated matrix metalloproteinase. These results implicate EP2 and EP4 receptors in the induction of VEGF expression and cell invasiveness by ET-1 and provide a mechanism by which ETAR/ET-1 can promote and interact with PGE2-dependent machinery to amplify its proangiogenic and invasive phenotype in ovarian carcinoma cells. Pharmacological blockade of ETAR can therefore represent an additional strategy to control PGE2 signaling, which has been associated with ovarian carcinoma progression.     

Phenotypic changes of p53, HER2, and FAS system in multiple normal tissues surrounding breast cancer

To determine whether phenotypic field changes occur in tissues adjacent to carcinoma, we assayed, by immunohistochemistry, the expression of HER-2, p53, Fas, and FasL in 72 breast cancers (BC) and multiple autologous peritumoral tissues (PTTs) sampled up to 5 cm distance and in 44 benign breast tumors (BBTs). About 5% and 3% of the PTTs and 4.5% and 6.8% of BBTs showed alterations in HER2 and p53 expression, respectively. Of interest, gene amplification was observed in 50% of HER2 positive PTTs, but not in any HER2 positive BBTs. Fas, highly expressed in BBTs and downregulated in BC, maintained its expression in PTTs, whereas FasL, usually negative in BBTs, was upregulated in BC as well as in the PTTs closest (1 cm) to the invasive lesion. Our data suggest that FasL could be a potential novel biomarker of transformation, which may identify, along with HER2 and p53, precursor lesions in a genetically altered breast tissue.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

Dysregulated miRNA biogenesis downstream of cellular stress and ALS‐causing mutations: a new mechanism for ALS

Interest in RNA dysfunction in amyotrophic lateral sclerosis (ALS) recently aroused upon discovering causative mutations in RNA‐binding protein genes. Here, we show that extensive down‐regulation of miRNA levels is a common molecular denominator for multiple forms of human ALS. We further demonstrate that pathogenic ALS‐causing mutations are sufficient to inhibit miRNA biogenesis at the Dicing step. Abnormalities of the stress response are involved in the pathogenesis of neurodegeneration, including ALS. Accordingly, we describe a novel mechanism for modulating microRNA biogenesis under stress, involving stress granule formation and re‐organization of DICER and AGO2 protein interactions with their partners. In line with this observation, enhancing DICER activity by a small molecule, enoxacin, is beneficial for neuromuscular function in two independent ALS mouse models. Characterizing miRNA biogenesis downstream of the stress response ties seemingly disparate pathways in neurodegeneration and further suggests that DICER and miRNAs affect neuronal integrity and are possible therapeutic targets.