Novel metal clusters isolated from blood are lethal to cancer cells.

Unfolding and subsequent aggregation of proteins is a common phenomenon that is linked to many human disorders. Misfolded hemoglobin is generally manifested in various autoimmune, infectious and inherited diseases. We isolated micrometer and submicrometer particles, termed proteons, from human and animal blood. Proteons lack nucleic acids but contain two major polypeptide populations with homology to the hemoglobin alpha-chain. Proteons form by reversible seeded aggregation of proteins around proteon nucleating centers (PNCs). PNCs are comprised of 1- to 2-nm metallic nanoclusters containing 40-300 atoms. Each milliliter of human blood contained approximately 7 x 10(13) PNCs and approximately 3 x 10(8) proteons. Exposure of isolated blood plasma to elevated temperatures increased the number of proteons. When an aliquot of this heated plasma was introduced into untreated plasma that was subsequently heated, the number of proteons further increased, reaching a maximum after a total of three such iterations. Small concentrations of PNCs were lethal to cultured cancer cells, whereas noncancerous cells were much less affected.

Phage matrix for isolation of glioma cell membrane proteins.

Cell-binding ligands for RG2 rat glioma were identified in our recent study from a library of peptides that are displayed as fusion molecules on phage particles. Here, one of the phage clones was used to affinity purify those cell membrane components to which the displayed peptides bind. This phage clone, displaying the ELRGDSLP peptide, was shown to recognize glioma cells specifically in comparison to control phage-expressing peptides of either similar or irrelevant sequences. Blocking experiments with synthetic RGDS peptide demonstrated that the phage-glioma cell recognition occurs via the RGD motif known to be present in many integrin-binding proteins. To form an affinity matrix that would bind to glioma cell membrane molecules, ELRGDSLP phage particles were cross-linked using dextran polymer. Whole cell lysate from RG2 rat glioma cells was passed through the matrix, resulting in the isolation of cell membrane components having strong affinity to the peptides on phage and molecules associated with those components. One of the isolated proteins was found to be CD44s, a cell surface adhesion molecule involved in glioma cell invasion and migration, which likely formed a complex with an RGD-binding integrin. Cell membrane proteins isolated with this innovative approach could be used for the design of cell-specific anticancer treatments.

Phage probes for malignant glial cells.

Early diagnosis and effective treatment of malignant gliomas, which are heterogeneous brain tumors with variable expression of cell surface markers, are inhibited by the lack of means to characterize and target tumor-selective molecules. To create molecular profiles for RG2 rat glioma cells, we used phage display technology, an approach capable of producing valuable ligands to unknown cell surface targets. The ligands were selected from libraries of peptides displayed as fusion molecules on phage particles. Modifications of the selection conditions resulted in identification of three distinctive families of peptide ligands for malignant glioma cells. The first family with V (D)/(G) L P (E)/(T) H(3) binding motif appeared to target a marker that is common for glioma cells, normal brain cells, and cells of non-brain origin. The second group of peptide-presented phage displayed D (T)/S/(L) T K consensus sequence and contained peptides with pronounced glioma-selective properties. Phage clones expressing peptides with E (L)/V/(S) R G D S motif were found in cell lysates and represented the third family of glioma-specific ligands. All peptides within this family contain the RGD amino acid sequence, which is known to bind to a number of integrins. Phage clones that belong to this family were internalized by RG2 glioma cells about 63-fold more efficiently than by astrocytes. The approach described could be applicable for accurate detection of glioma expression patterns in individual tumors. Such patterns could be beneficial in the design of effective combinations of drugs for anti-glioma treatments.

Peptide biosensor for recognition of cross-species cell surface markers.

Biosensors based on phage display-derived peptides as biorecognition molecules were used for the detection of cell surface cross-species markers in tissue homogenates. The peptide selected for murine myofibers was immobilized onto the surface of an acoustic wave sensor by biotin-streptavidin coupling. To detect peptide-receptor interaction, the sensors were exposed to muscle and control (kidney, liver, brain) tissue homogenates. The sensor showed a strong response to murine muscle. The amplitudes of the responses to the feline muscle homogenates were lower compared to those of the murine muscle, while the same K(d) indicated that the peptide has cross-species affinity. In contrast, murine kidney, liver and brain homogenates produced insignificant responses. Specificity of the sensor was shown in a blocking experiment, as reduced signal was detected when muscle preparations were preincubated with free peptide. Additionally, when muscle-specific peptide was replaced with two different random control peptides, the sensors produced no response to murine muscle. Suitability of peptide ligands for a variety of species can be evaluated using this technology.

Targeting peptides for microglia identified via phage display.

Screening with a 7-mer phage display peptide library, a panel of cell-targeting peptides for the murine microglial cell line, EOC 20, was recognized. A number of similar, but not identical, sets of sequences representing more than 75% of all the cell line-binding clones were identified. Comparative analysis indicated that motif S/(T) F T/(X) Y W is present in the vast majority of the binding sequences. The selectivity and specificity of the dominant peptide sequence identified for microglia was confirmed using both phage displaying the peptide and the synthetic peptide alone.