Expression in cowpea seedlings of chimeric transgenes after electroporation into seed-derived embryos

Summary Cowpea (Vigna unguiculata Walp) embryos mechanically isolated from mature seeds and incubated in the presence of plasmid DNA harboring chimeric gus genes were shown to germinate into seedlings expressing -glucuronidase activity in a variety of tissues, including the apical meristem. Embryo electroporation in the presence of DNA and protectants such as spermine and Lipofectin^TM increased both the proportion of embryo-derived seedlings expressing the chimeric gene and the level of gene expression. Microscopic observations of thin sections showed that the blue crystals representing the end product of transgene activity on X-glu were exclusively located inside the treated cells. Histological localization of the blue dye crystals varied with the promoter used to drive the transgene.     

Monoclonal antibodies to Escherichia coli ribosomal proteins L9 and L10. Effects on ribosome function and localization of L9 on the surface of the 50 S ribosomal subunit

Monoclonal antibodies against Escherichia coli ribosomal proteins L9 and L10 were obtained and their specificity confirmed by Western blot analysis of total ribosomal protein. This was particularly important for the L9 antibody, since the immunizing antigen mixture contained predominantly L11. Each antibody recognized both 70 S ribosomes and 50 S subunits. Affinity-purified antibodies were tested for their effect on in vitro assays of ribosome function. Anti-L10 and anti-L9 inhibited poly(U)-directed polyphenylalanine synthesis almost completely. The antibodies had no effect on subunit association or dissociation and neither antibody inhibited peptidyltransferase activity. Both antibodies inhibited the binding of the ternary complex that consisted of aminoacyl-tRNA, guanylyl beta, gamma-methylenediphosphonate, and elongation factor Tu, and the binding of elongation factor G to the ribosome. The intact antibodies were more potent inhibitors than the Fab fragments. In contrast to the previously established location of L10 at the base of the L7/L12 stalk near the factor-binding site, the site of anti-L9 binding to 50 S subunits was shown by immune electron microscopy to be on the L1 lateral protuberance opposite the L7/L12 stalk as viewed in the quasisymmetric projection. The inhibition of factor binding by both antibodies, although consistent with established properties of L10 in the ribosome, suggests a long range effect on subunit structure that is triggered by the binding of anti-L9.     

Synthesis of DNA interactive C3-trans-cinnamide linked β-carboline conjugates as potential cytotoxic and DNA topoisomerase I inhibitors

A series of new C3-trans-cinnamide linked β-carboline conjugates has been synthesized by coupling between various β-carboline amines and substituted cinnamic acids. Evaluation of their anti-proliferative activity against a panel of selected human cancer cell lines such as A549 (lung cancer), MCF-7 (breast cancer), B16 (melanoma), HeLa (cervical cancer) and a normal cell line NIH3T3 (mouse embryonic fibroblast cell line), suggested that the newly designed conjugates are considerably active against all the tested cancer cell lines with IC₅₀ values 13–45?nM. Moreover, the conjugates 8v and 8x were the most active against MCF-7 cells (14.05?nM and 13.84?nM respectively) and also even potent on other cell lines tested. Further, detailed investigations such as cell cycle analysis, apoptosis induction study, topoisomerase I inhibition assay, DNA binding affinity and docking studies revealed that these new conjugates are DNA interactive topoisomerase I inhibitors.     

Structure of ebenone,a possible biogenetic precursor of elliptinone,from Diospyros ebenum

The isolation and determination of the structure of ebenone, a new biogenetically significant β-naphthyl-1,4-naphthaquinone derivative, from Diospyros ebenum is reported.     

SeMoP: a new computational strategy for the unrestricted search for modified peptides using LC-MS/MS data

A novel computational approach, termed Search for Modified Peptides (SeMoP), for the unrestricted discovery and verification of peptide modifications in shotgun proteomic experiments using low resolution ion trap MS/MS spectra is presented. Various peptide modifications, including post-translational modifications, sequence polymorphisms, as well as sample handling-induced changes, can be identified using this approach. SeMoP utilizes a three-step strategy: (1) a standard database search to identify proteins in a sample; (2) an unrestricted search for modifications using a newly developed algorithm; and (3) a second standard database search targeted to specific modifications found using the unrestricted search. This targeted approach provides verification of discovered modifications and, due to increased sensitivity, a general increase in the number of peptides with the specific modification. The feasibility of the overall strategy has been first demonstrated in the analysis of 65 plasma proteins. Various sample handling induced modifications, such as beta-elimination of disulfide bridges and pyrocarbamidomethylation, as well as biologically induced modifications, such as phosphorylation and methylation, have been detected. A subsequent targeted Sequest search has been used to verify selected modifications, and a 4-fold increase in the number of modified peptides was obtained. In a second application, 1367 proteins of a cervical cancer cell line were processed, leading to detection of several novel amino acid substitutions. By conducting the search against a database of peptides derived from proteins with decoy sequences, a false discovery rate of less than 5% for the unrestricted search resulted. SeMoP is shown to be an effective and easily implemented approach for the discovery and verification of peptide modifications.     

Host response profile of human brain proteome in toxoplasma encephalitis co-infected with HIV

Background

Toxoplasma encephalitis is caused by the opportunistic protozoan parasite Toxoplasma gondii. Primary infection with T. gondii in immunocompetent individuals remains largely asymptomatic. In contrast, in immunocompromised individuals, reactivation of the parasite results in severe complications and mortality. Molecular changes at the protein level in the host central nervous system and proteins associated with pathogenesis of toxoplasma encephalitis are largely unexplored. We used a global quantitative proteomic strategy to identify differentially regulated proteins and affected molecular networks in the human host during T. gondii infection with HIV co-infection.

Results

We identified 3,496 proteins out of which 607 proteins were differentially expressed (≥1.5-fold) when frontal lobe of the brain from patients diagnosed with toxoplasma encephalitis was compared to control brain tissues. We validated differential expression of 3 proteins through immunohistochemistry, which was confirmed to be consistent with mass spectrometry analysis. Pathway analysis of differentially expressed proteins indicated deregulation of several pathways involved in antigen processing, immune response, neuronal growth, neurotransmitter transport and energy metabolism.

Conclusions

Global quantitative proteomic approach adopted in this study generated a comparative proteome profile of brain tissues from toxoplasma encephalitis patients co-infected with HIV. Differentially expressed proteins include previously reported and several new proteins in the context of T. gondii and HIV infection, which can be further investigated. Molecular pathways identified to be associated with the disease should enhance our understanding of pathogenesis in toxoplasma encephalitis.

Electronic supplementary material

The online version of this article (doi:10.1186/1559-0275-11-39) contains supplementary material, which is available to authorized users.