Connexin 40 promoter-based enrichment of embryonic stem cell-derived cardiovascular progenitor cells

Background: Pluripotent embryonic stem (ES) cells that can differentiate into functional cardiomyocytes as well as vascular cells in cell culture may open the door to cardiovascular cell transplantation. However, the percentage of ES cells in embryoid bodies (EBs) which spontaneously undergo cardiovascular differentiation is low (< 10%), making strategies for their specific labeling and purification indispensable. Methods: The human connexin 40 (Cx40) promoter was isolated and cloned in the vector pEGFP. The specificity of the construct was initially assessed in Xenopus embryos injected with Cx40-EGFP plasmid DNA. Stable Cx40-EGFP ES cell clones were differentiated and fluorescent cells were enriched manually as well as via fluorescence-activated cell sorting. Characterization of these cells was performed with respect to spontaneous beating as well as via RT-PCRs and immunofluorescent stainings. Results: Cx40-EGFP reporter plasmid injection led to EGFP fluorescence specifically in the abdominal aorta of frog tadpoles. After crude manual enrichment of highly Cx40-EGFP- positive EBs, the appearance of cardiac and vascular structures was increased approximately 3-fold. Immuno fluorescent stainings showed EGFP expression exclusively in vascular-like structures simultaneously expressing von Willebrand factor and in formerly beating areas expressing alpha-actinin. Cx40-EGFP-expressing EBs revealed significantly higher numbers of beating cardiomyocytes and vascular-like structures. Semiquantitative RT-PCRs confirmed an enhanced cardiovascular differentiation as shown for the cardiac markers Nkx2.5 and MLC2v, as well as the endothelial marker vascular endothelial cadherin. Conclusions: Our work shows the feasibility of specific labeling and purification of cardiovascular progenitor cells from differentiating EBs based on the Cx40 promoter. We provide proof of principle that the deleted CD4 (Delta CD4) surface marker-based method for magnetic cell sorting developed by our group will be ideally suitable for transference to this promoter. Copyright (c) 2008 S. Karger AG, Basel

Energy transfer from retinal to amino acids — a time-resolved study of the ultraviolet emission of bacteriorhodopsin

Two-step excitation of retinal in bacteriorhodopsin by visible light is followed by an energy transfer to amino acids that is seen as fluorescent emission around 350 nm. The fluorescence spectrum obtained after two-step excitation (2 × 527 nm) differs from the fluorescence spectrum obtained after one-step ultraviolet excitation (263.5 nm) by a strongly quenched emission with a fluorescence lifetime of 10 ± 5 ps and a smaller spectral width. The two-step absorption process presumably selects tryptophan residues which strongly couple to the retinal chromophore

Early picosecond events in the photo cycle of Bacteriorhodopsin

The primary processes of the photochemical cycle of light-adapted bacteriorhodopsin (BR) were studied by various experimental techniques with a time resolution of 5 × 10-13 s. The following results were obtained. (a) After optical excitation the first excited singlet state S1 of bacteriorhodopsin is observed via its fluorescence and absorption properties. The population of the excited singlet state decays with a lifetime τ1 of ~0.7 ps (430 ± 50 fs) (52). (b) With the same time constant the first ground-state intermediate J builds up. Its absorption spectrum is red-shifted relative to the spectrum of BR by ~30 nm. (c) The second photoproduct K, which appears with a time constant of τ2 = 5 ps shows a red-shift of 20 nm, relative to the peak of BR. Its absorption remains constant for the observation time of 300 ps. (d) Upon suspending bacteriorhodopsin in D2O and deuterating the retinal Schiff base at its nitrogen (lysine 216), the same photoproducts J and K are observed. The relaxation time constants τ1 and τ2 remain unchanged upon deuteration within the experimental accuracy of 20%

Optical picosecond studies of bacteriorhodopsin containing a sterically fixed retinal

The photochemical behaviour of an analogous bacteriorhodopsin (9,12-Ph-BR) which contains the sterically fixed 9,12-phenylretinal has been investigated with picosecond spectroscopy. The following results have been obtained. No ground-state intermediate photoproduct is found in agreement with the previous observation that 9,12-Ph-BR does not exhibit proton pumping under illumination. The excited singlet state has a lifetime of τS = 10 ± 2 ps. This lifetime agrees favourably with the value calculated from the radiative lifetime τrad = 6.2 ns and the fluorescence quantum efficiency of 1.2·10−3. Excited-state absorption occurs which results in fluorescence in the ultraviolet region. These various observations differ drastically from the corresponding findings on bacteriorhodopsin. Most important for an understanding of the differences is the fact that 9,12-phenylretinal does not isomerize in the protein's binding site in contrast to retinal. Our data therefore suggest that the formation of the intermediate K observed in bacteriorhodopsin is accompanied by the all-trans to 13-cis isomerization

Instantaneous Bethe-Salpeter equation: improved analytical solution

Studying the Bethe-Salpeter formalism for interactions instantaneous in the rest frame of the bound states described, we show that, for bound-state constituents of arbitrary masses, the mass of the ground state of a given spin may be calculated almost entirely analytically with high accuracy, without the (numerical) diagonalization of the matrix representation obtained by expansion of the solutions over a suitable set of basis states.Comment: 7 page

Discrete Spectra of Semirelativistic Hamiltonians

We review various attempts to localize the discrete spectra of semirelativistic Hamiltonians of the form H = \beta \sqrt{m^2 + p^2} + V(r) (w.l.o.g. in three spatial dimensions) as entering, for instance, in the spinless Salpeter equation. Every Hamiltonian in this class of operators consists of the relativistic kinetic energy \beta \sqrt{m^2 + p^2} (where \beta > 0 allows for the possibility of more than one particles of mass m) and a spherically symmetric attractive potential V(r), r = |x|. In general, accurate eigenvalues of a nonlocal Hamiltonian operator can only be found by the use of a numerical approximation procedure. Our main emphasis, however, is on the derivation of rigorous semi-analytical expressions for both upper and lower bounds to the energy levels of such operators. We compare the bounds obtained within different approaches and present relationships existing between the bounds.Comment: 21 pages, 3 figure

Energy bounds for the spinless Salpeter equation: harmonic oscillator

We study the eigenvalues E_{n\ell} of the Salpeter Hamiltonian H = \beta\sqrt(m^2 + p^2) + vr^2, v>0, \beta > 0, in three dimensions. By using geometrical arguments we show that, for suitable values of P, here provided, the simple semi-classical formula E = min_{r > 0} {v(P/r)^2 + \beta\sqrt(m^2 + r^2)} provides both upper and lower energy bounds for all the eigenvalues of the problem.Comment: 8 pages, 1 figur