Use of SPR to Study the Interaction of G7-18NATE Peptide with the Grb7-SH2 Domain

Surface plasmon resonance (SPR) is a useful biosensor technique for the study of biomolecular interactions, with the potential for high-throughput screening of ligand interactions with drug targets. The key to its successful use, however, is in the appropriate design of the experiment, including the mode of immobilization to the biosensor chip. We report an investigation of the use of SPR for measuring the affinity of the G7-18NATE peptide ligand for its Grb7-SH2 domain target involved in the migratory and proliferative potential of cancer cells. Previous studies have shown that the cyclic non-phosphorylated peptide, G7-18NATE, inhibits Grb7 interactions with upstream binding partners and is able to inhibit both cell migration and proliferation of cancer cells. We report the synthesis of a biotinylated G7-18NATE covalently attached to a linker (G7-18NATE-ASASASK-Biotin) and compare its interaction with the Grb7-SH2 domain by SPR using three different immobilization strategies; immobilisation of the peptide via streptavidin, immobilization of glutathione S-transferase (GST)-Grb7-SH2 domain via anti-GST antibody, and immobilization of biotinylated Grb7-SH2 domain via streptavidin. This revealed that sensorgrams free from non-specific binding and displaying simple kinetics were most readily achieved by immobilising the protein rather than the peptide, in spite of the lower response associated with this method. K _D values of ~300 μM were determined for both strategies at pH 7.4. This compared with a K _D value of 4.4 μM at pH 6 demonstrating the importance of pH on this interaction. Overall, the immobilised protein systems are most suitable for future comparative screening efforts using SPR.     

MFASAT: a new alphoid DNA sequence isolated from Macaca fascicularis (Cercopithecidae, Primates).

A new highly repeated DNA fragment isolated from Macaca fascicularis (MFASAT) is described. Our findings obtained by sequencing, Southern blot analysis, and fluorescent in situ hybridization (FISH) on metaphasic chromosomes strongly suggest that MFASAT can be considered as a member of the alphoid DNA family characteristic of Old World monkeys. The chromosomal localization of MFASAT, obtained by FISH, showed that this alphoid DNA is present in the peri-centromeric area of all the chromosomes. MFASAT showed a high degree of conservation when compared, by sequence alignment, to other Macaca species and Papio papio as expected for species with considerable genome conservation. A low degree of homology has been found comparing M. fascicularis alphoid DNA with a more distantly related Cercopithecidae species such as Cercopithecus aethiops.     

First survey of the wheat chromosome 5A composition through a next generation sequencing approach

Wheat is one of the world's most important crops and is characterized by a large polyploid genome. One way to reduce genome complexity is to isolate single chromosomes using flow cytometry. Low coverage DNA sequencing can provide a snapshot of individual chromosomes, allowing a fast characterization of their main features and comparison with other genomes. We used massively parallel 454 pyrosequencing to obtain a 2x coverage of wheat chromosome 5A. The resulting sequence assembly was used to identify TEs, genes and miRNAs, as well as to infer a virtual gene order based on the synteny with other grass genomes. Repetitive elements account for more than 75% of the genome. Gene content was estimated considering non-redundant reads showing at least one match to ESTs or proteins. The results indicate that the coding fraction represents 1.08% and 1.3% of the short and long arm respectively, projecting the number of genes of the whole chromosome to approximately 5,000. 195 candidate miRNA precursors belonging to 16 miRNA families were identified. The 5A genes were used to search for syntenic relationships between grass genomes. The short arm is closely related to Brachypodium chromosome 4, sorghum chromosome 8 and rice chromosome 12; the long arm to regions of Brachypodium chromosomes 4 and 1, sorghum chromosomes 1 and 2 and rice chromosomes 9 and 3. From these similarities it was possible to infer the virtual gene order of 392 (5AS) and 1,480 (5AL) genes of chromosome 5A, which was compared to, and found to be largely congruent with the available physical map of this chromosome.     

Persistent response to pneumococcal vaccine in individuals supplemented with a novel water soluble extract of Uncaria tomentosa, C-Med-100

A human intervention study was carried out using male volunteers attending a General Practice Clinic in New York City involving comparison of individuals supplemented with 350 mg x 2 C-Med-100 daily dose for two months with untreated controls for their abilities to respond to a 23 valent pneumococcal vaccine. C-Med-100 is a novel nutraceutical extract from the South American plant Uncaria tomentosa or Cat's Claw which is known to possess immune enhancing and antiinflammatory properties in animals. There were no toxic side effects observed as judged by medical examination, clinical chemistry and blood cell analysis. However, statistically significant immune enhancement for the individuals on C-Med-100 supplement was observed by (i) an elevation in the lymphocyte/neutrophil ratios of peripheral blood and (ii) a reduced decay in the 12 serotype antibody titer responses to pneumococcal vaccination at 5 months.     

Unscheduled DNA synthesis induced by N-acetoxy-2-acetylaminofluorene is not sensitive to regulation by ADP-ribosyl transferase

We have directly compared in resting human mononuclear leukocytes the DNA repair effects caused by ADP-ribosyl transferase (ADPRT) activity following DNA damage induction by gamma radiation, UV radiation, ethylene oxide (EO) and N-acetoxy-2-acetylaminofluorene (NA-AAF). The presence of inhibitors of ADPRT during the quantitation of unscheduled DNA synthesis (UDS) resulted in about a 2-fold increase of UDS when induced by gamma radiation, UV radiation or EO. The stimulation of UDS by EO, UV- or gamma-radiation in the presence of an ADPRT inhibitor was equally strong whether 1 mM or 10 mM hydroxyurea was used to suppress scheduled DNA synthesis. The level of NA-AAF induced UDS was not affected by inhibitors of ADPRT. In addition, direct estimation of ADPRT activity revealed that at doses giving maximal UDS, NA-AAF damage did not induce a measurable enzymatic activity whereas gamma-radiation, UV radiation and EO all showed a significant dose response increase. We have interpreted our data to mean that NA-AAF induced UDS estimates DNA repair relating mainly to DNA lesions that are recognized with difficulty, and hence, the rate of endonuclease-induced DNA strand break accumulation is not sufficient to allow a stimulation of ADPRT and affect the quantitation of UDS.