The rate of retinal photoisomerization in wild-type bacteriorhodopsin (wt bR) is compared with that in a number of mutants in which a positively charged (Arg(82)), a negatively charged (Asp(85) or Asp(212)), or neutral hydrogen bonding (Asp(115) or Tyr(185)) amino acid residue known to be functionally important within the retinal cavity is replaced by a neutral, non-hydrogen bonding one. Only the replacements of the charged residues reduced the photoisomerization rate of the 13-cis and all-trans isomers present in these mutants by factors of approximately 1/4 and approximately 1/20, respectively. Retinal photo- and thermal isomerization catalysis and selectivity in wt bR by charged residues is discussed in terms of the known protein structure, the valence-bond wave functions of the ground and excited state of the retinal, and the electrostatic stabilization interactions within the retinal cavity.

No abstract

The dynamics of all-trans and 13-cis retinal protonated Schiff base (RPSB) were studied in different solvents by means of picosecond transient spectroscopy. The decay time of the excited state absorption was found to be wavelength dependent due to the contribution of the faster decay of stimulated emission. The stimulated emission has a lifetime of a 2.5−4 ps while the excited state absorption decay is biexponential with lifetimes of 2.5−4 and 10−12 ps. The fluorescence quantum yield is strongly temperature dependent, but viscosity has a small effect on both excited-state lifetime and fluorescence quantum yield. This leads to the conclusion that there is a ∼600 cm-1 barrier in the excited-state which results from intramolecular electronic factors and not from the solvent viscosity. The comparison of these results with those for the retinal in rhodopsin and bacteriorhodopsin is discussed in terms of the protein catalysis for the retinal photoisomerization.

The differentiation of stem cells into smooth muscle cells (SMCs) plays an important role in vascular development and remodeling. In addition, stem cells represent a potential source of SMCs for regenerative medicine applications such as constructing vascular grafts. Previous studies have suggested that various biochemical factors, including transforming growth factor-β (TGF-β) and the Notch pathway, may play important roles in vascular differentiation. However, the interactions of these two signaling pathways in the differentiation of bone marrow mesenchymal stem cells (MSCs) have not been clearly defined. In this study, we profiled the gene expression in MSCs in response to TGF-β, and showed that TGF-β induced Notch ligand Jagged 1 (JAG1) and SMC markers, including smooth muscle α-actin (ACTA2), calponin 1 (CNN1), and myocardin (MYOCD), which were dependent on the activation of SMAD3 and Rho kinase. In addition, knocking down JAG1 expression partially blocked ACTA2 and CNN1 expression and completely blocked MYOCD expression, suggesting that JAG1 plays an important role in TGF-β-induced expression of SMC markers. On the other hand, the activation of Notch signaling induced the expression of SMC markers in MSCs and human embryonic stem cells (hESCs). Notch activation in hESCs also resulted in an increase of neural markers and a decrease of endothelial markers. These results suggest that Notch signaling mediates TGF-β regulation of MSC differentiation and that Notch signaling induces the differentiation of MSCs and hESCs into SMCs, which represents a novel mechanism involved in stem cell differentiation.

The recently developed DNA microarray technology provides a powerful and efficient tool to rapidly compare the differential expression of a large number of genes. Using the DNA microarray approach, we investigated gene expression profiles in cultured human aortic endothelial cells (HAECs) in response to 24 h of laminar shear stress at 12 dyn/cm(2). This relatively long-term shearing of cultured HAECs led to the modulation of the expression of a number of genes. Several genes related to inflammation and EC proliferation were downregulated, suggesting that 24-h shearing may keep ECs in a relatively noninflammatory and nonproliferative state compared with static cells. Some genes were significantly upregulated by the 24-h shear stress; these includes genes involved in EC survival and angiogenesis (Tie2 and Flk-1) and vascular remodeling (matrix metalloproteinase 1). These results provide information on the profile of gene expression in shear-adapted ECs, which is the case for the native ECs in the straight part of the aorta in vivo.

Classical mechanisms of heterotrimeric G-protein signaling are observed to function in regulation of the transcriptome. Conversely, many theoretical regulatory modes of the G-protein are not manifested in the transcriptomes we investigate.A new mechanism of G-protein signaling is revealed, in which the β subunit regulates gene expression identically in the presence or absence of the α subunit.We find evidence of cross-talk between G-protein-mediated and hormone-mediated transcriptional regulation.We find evidence of system specificity in G-protein signaling.

The quantum yield of photoisomerization and the energy content of the K intermediate in the photocycle of bacteriorhodopsin and its mutants D85N, R82Q, and D212N and deionized blue bR were measured. Transient optical absorption and photoacoustic spectroscopy with excitation using 400 fs laser pulse were combined to obtain results. The spectroscopic characteristics of the excited state, the J and K intermediates in the photocycle of the mutants, and deionized blue bR were determined. The presence of both 13-cis and all-trans isomers in the ground state of light-adapted D85N, R82Q, and D212N and deionized blue bR makes extraction of the quantum yield for each isomer difficult. Thus, only average values of the quantum yield for these samples were determined. The replacement of charged groups in the vicinity of the retinal Schiff base was found to decrease the rate of the photoisomerization by up to 30 times, but with no signficant change in either the apparent quantum yield of the photoisomerization or the energy stored in the K intermediate. The results are discussed in terms of the different models for the excited and ground state potential surfaces of the retinal configuration in bacteriorhodopsin.

No abstract