The noninvasive analysis of living cells grown on 3-dimensional scaffold materials is a key point in tissue engineering. In this work we show the capability of Raman spectroscopy for use as a noninvasive method to distinguish cells at different stages of the cell cycle and living cells from dead cells. The spectral differences between cells in different stages of the cell cycle are characterized mainly by variations in DNA vibrations at 782, 788, and 1095 cm(-1). The Raman spectrum of dead human lung derived (A549 line) cells indicates the breakdown of both phosphodiester bonds and DNA bases. The most sensitive peak for identifying dead cells is the 788 cm(-1) peak corresponding to DNA Obond;Pbond;O backbone stretching. The magnitude of this peak is reduced by 80% in the spectrum of dead cells. Changes in protein peaks suggest significant conformational changes; for example, the magnitude of the 1231 cm(-1) peak assigned to random coils is reduced by 63% for dead cells. The sharp peak of phenylalanine at 1005 cm(-1) drops to half, indicating a decrease of stable proteins associated with cell death. The differences in the 1190-1385 cm(-1) spectral region also suggest a decrease in the amount of nucleic acids and proteins. Using curve fitting, we quantify these spectral differences that can be used as markers of cell death.

We report the first Raman spectra of individual living and dead cells (MLE-12 line) cultured on bioinert standard poly-L-lysine coated fused silica and on bioactive 45S5 Bioglass®measured at 785 nm laser excitation. At this excitation wavelength no damage was induced to the cells even after 40 minutes irradiation at 115 mW power, as indicated by cell morphology observation and trypan blue viability test. We show that shorter wavelength lasers, 488 nm and 514 nm, cannot be used because they induce damage to the cells at very low laser powers (5 mW) and short irradiation times (5–20 minutes). The most important differences between the spectra of living and dead cells are in the 1530–1700 cm−1range, where the dead cells have strong peaks at 1578 cm−1and 1607 cm−1. Other differences occur around the DNA peak at 1094 cm−1. Our study establishes the feasibility of using the 785 nm laser for anin situreal-time non-invasive method to follow biological events (proliferation, differentiation, cell death, etc.) within individual cells cultured on bioactive scaffolds in their physiologic environment over long periods of time.

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We investigated the use of Raman microspectroscopy to monitor the molecular changes in human lung carcinoma epithelial cells (A549) when cell death was induced by a toxic chemical. We treated A549 cells with 100 microM Triton X-100 and carried out Raman microspectroscopy measurements in parallel with cell viability and DNA integrity assays at time points of 0, 24, 48, and 72 hours. We found that the important biochemical changes taking place during cell death, such as the degradation of proteins, DNA breakdown, and the formation of lipid vesicles, can be detected with Raman microspectroscopy. A decrease in the intensity of the O-P-O stretching Raman peak corresponding to the DNA molecule phosphate-sugar backbone at 788 cm(-1) indicated DNA disintegration, an observation which was confirmed by DNA integrity analysis. We also found a decrease in the intensity of the Raman peaks corresponding to proteins (1005 cm(-1), 1342 cm(-1)) and an increase in the concentration of lipids (1660 cm(-1), 1303 cm(-1)). These changes are the effects of the complex molecular mechanisms during the induction of cell death, such as protein cleavage due to the activation of caspases, followed by DNA fragmentation.

STAT proteins have the function of signaling from the cell membrane into the nucleus, where they regulate gene transcription. Latent mammalian STAT proteins can form dimers in the cytoplasm even before receptor-mediated activation by specific tyrosine phosphorylation. Here we describe the 3.21-Å crystal structure of an unphosphorylated STAT5a homodimer lacking the N-terminal domain as well as the C-terminal transactivation domain. The overall structure of this fragment is very similar to phosphorylated STATs. However, important differences exist in the dimerization mode. Although the interface between phosphorylated STATs is mediated by their Src-homology 2 domains, the unphosphorylated STAT5a fragment dimerizes in a completely different manner via interactions between their ␤-barrel and four-helix bundle domains. The STAT4 N-terminal domain dimer can be docked onto this STAT5a core fragment dimer based on shape and charge complementarities. The separation of the dimeric arrangement, taking place upon activation and nuclear translocation of STAT5a, is demonstrated by fluorescence resonance energy transfer experiments in living cells. STAT4 (signal transducer and activator of transcription) proteins mediate the signaling of cytokines and a number of growth factors from the receptors of these extracellular signaling molecules to the cell nucleus. Dependent on the receptor type STATs are specifically phosphorylated by receptor-associated Janus kinases, receptor tyrosine kinases, or cytoplasmic tyrosine kinases (1). The phosphorylated STAT molecules dimerize by reciprocal binding of their SH2 domains to the phosphotyrosine residues. These dimeric STATs translocate into the nucleus, bind to specific DNA sequences, and regulate the transcription of their target genes.Seven mammalian STATs have been identified. Their structural organization is known in molecular detail from several crystal structures. At the N terminus they contain a helical domain, which mediates cooperative binding of STATs to sequential DNA binding sites (2). The structures of the phosphorylated STAT1 and STAT3 core fragments, lacking the N-terminal domain as well as the C-terminal transactivation domain, were solved in complex with DNA (3, 4). They consist of an N-terminal large four-helix bundle, a central IgG-like domain, which constitutes the actual DNA binding domain, a helical, so-called linker domain, and the SH2 domain. The phosphotyrosine residue is located in all mammalian STATs within the 30 amino acids C-terminal to the SH2 domain. In most mammalian STATs the C-terminal part is constituted by a mostly disordered transactivation domain, which mediates the interactions of STATs with other components of the transcription machinery. Three-dimensional information for this domain so far has only been obtained from the complex of a short fragment of the STAT6 transactivation domain with the PAS-B domain of the nuclear receptor coactivator 1 (5).STAT3 homodimers and STAT3-STAT1 complexes have been coimmunoprecipitated from untreated cells (6, 7)...

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The cholecystokinin (CCK) receptor-1 (CCK1R) is a G protein-coupled receptor, which mediates important central and peripheral cholecystokinin actions. Our aim was to progress in mapping of the CCK1R binding site by identifying residues that interact with the methionine and phenylalanine residues of the C-terminal moiety of CCK because these are crucial for its binding and These new and important insights will serve to better understand the activation process of CCK1R and to design or optimize ligands.