Adult-derived stem cells and their potential for use in tissue repair and molecular medicine

This report reviews three categories of precursor cells present within adults. The first category of precursor cell, the epiblast-like stem cell, has the potential of forming cells from all three embryonic germ layer lineages, e.g., ectoderm, mesoderm, and endoderm. The second category of precursor cell, the germ layer lineage stem cell, consists of three separate cells. Each of the three cells is committed to form cells limited to a specific embryonic germ layer lineage. Thus the second category consists of germ layer lineage ectodermal stem cells, germ layer lineage mesodermal stem cells, and germ layer lineage endodermal stem cells. The third category of precursor cells, progenitor cells, contains a multitude of cells. These cells are committed to form specific cell and tissue types and are the immediate precursors to the differentiated cells and tissues of the adult. The three categories of precursor cells can be readily isolated from adult tissues. They can be distinguished from each other based on their size, growth in cell culture, expressed genes, cell surface markers, and potential for differentiation. This report also discusses new findings. These findings include the karyotypic analysis of germ layer lineage stem cells; the appearance of dopaminergic neurons after implantation of naive adult pluripotent stem cells into a 6-hydroxydopamine-lesioned Parkinson's model; and the use of adult stem cells as transport mechanisms for exogenous genetic material. We conclude by discussing the potential roles of adult-derived precursor cells as building blocks for tissue repair and as delivery vehicles for molecular medicine.     

New class of competitive inhibitor of bacterial histidine kinases

Bacterial histidine kinases have been proposed as targets for the discovery of new antibiotics, yet few specific inhibitors of bacterial histidine kinases have been reported. We report here a novel thienopyridine (TEP) compound that inhibits bacterial histidine kinases competitively with respect to ATP but does not comparably inhibit mammalian serine/threonine kinases. Although it partitions into membranes and does not inhibit the growth of bacterial or mammalian cells, TEP could serve as a starting compound for a new class of histidine kinase inhibitors with antibacterial activity.     

A unified view of the role of electrostatic interactions in modulating the gating of Cys loop receptors

In the Cys loop superfamily of ligand-gated ion channels, a global conformational change, initiated by agonist binding, results in channel opening and the passage of ions across the cell membrane. The detailed mechanism of channel gating is a subject that has lent itself to both structural and electrophysiological studies. Here we defined a gating interface that incorporates elements from the ligand binding domain and transmembrane domain previously reported as integral to proper channel gating. An overall analysis of charged residues within the gating interface across the entire superfamily showed a conserved charging pattern, although no specific interacting ion pairs were conserved. We utilized a combination of conventional mutagenesis and the high precision methodology of unnatural amino acid incorporation to study extensively the gating interface of the mouse muscle nicotinic acetylcholine receptor. We found that charge reversal, charge neutralization, and charge introduction at the gating interface are often well tolerated. Furthermore, based on our data and a reexamination of previously reported data on gamma-aminobutyric acid, type A, and glycine receptors, we concluded that the overall charging pattern of the gating interface, and not any specific pairwise electrostatic interactions, controls the gating process in the Cys loop superfamily.     

Site-specific disulfide capture of agonist and antagonist peptides on the C5a receptor

The manner by which peptidic ligands bind and activate their corresponding G-protein-coupled receptors is not well understood. One of the better characterized peptidic ligands is the chemotactic cytokine complement factor 5a (C5a), a 74-amino acid helical bundle. Previous studies showed 6-mer peptide analogs derived from the C terminus of the C5a ligand can bind to C5aR (Kd values approximately 0.1-1 microm) and either agonize or antagonize the receptor (Gerber, B. O., Meng, E. C., Dotsch, V., Baranski, T. J., and Bourne, H. R. (2001) J. Biol. Chem. 276, 3394-3400). Here, we provide direct biochemical data using disulfide trapping to support a model that these peptides bind within a transmembrane helical triad formed by alpha-helices III, VI, and VII. We show that the three amino acids on the C terminus of the peptide analogs bind too weakly to exert a functional effect themselves. However, when a cysteine residue is placed on their N terminus they can be trapped by disulfide interchange to specific cysteines in helix III and VI and not to other cysteines, engineered into the C5aR. The trapped peptides function as agonists or partial antagonists, similar to the non-covalent parents from which they were derived. These data help to further refine the binding mode for C5a to the C5aR and suggest an approach and a binding site that may be applicable to studying other peptide binding receptors.     

Identification of a region of fast skeletal troponin T required for stabilization of the coiled-coil formation with troponin I

We have previously identified evolutionarily conserved heptad hydrophobic repeat (HR) domains in all isoprotein members of troponin T (TnT) and troponin I (TnI), two subunits of the Ca(2+)-regulatory troponin complex. Our suggestion that the HR domains are involved in the formation of a coiled-coil heterodimer of TnT and TnI has been recently confirmed by the crystal structure of the core domain of the human cardiac troponin complex. Here we studied a series of recombinant deletion mutants of the fast skeletal TnT to determine the minimal sequence required for stable coiled-coil formation with the HR domain of the fast skeletal TnI. Using circular dichroism spectroscopy, we measured the alpha helical content of the coiled-coil formed by the various TnT peptides with TnI HR domain. Sedimentation equilibrium experiments confirmed that the individual peptides of TnT were monomeric but formed heterodimers when mixed with HR domain of TnI. Isothermal titration calorimetry was then used to directly measure the affinity of the TnT peptides for the TnI HR domain. Surprisingly we found that the HR regions alone of the fast skeletal TnT and TnI, as defined earlier, were insufficient to form a coiled-coil. Furthermore we showed that an additional 14 amino acid residues N-terminal to the conserved HR region (TnT residues 165-178) are essential for the stable coiled-coil formation. We discuss the implication of our finding in the fast skeletal troponin isoform in the light of the crystal structure of the cardiac isoform.     

Photobiomodulation directly benefits primary neurons functionally inactivated by toxins: role of cytochrome c oxidase

Far red and near infrared (NIR) light promotes wound healing, but the mechanism is poorly understood. Our previous studies using 670 nm light-emitting diode (LED) arrays suggest that cytochrome c oxidase, a photoacceptor in the NIR range, plays an important role in therapeutic photobiomodulation. If this is true, then an irreversible inhibitor of cytochrome c oxidase, potassium cyanide (KCN), should compete with LED and reduce its beneficial effects. This hypothesis was tested on primary cultured neurons. LED treatment partially restored enzyme activity blocked by 10-100 microm KCN. It significantly reduced neuronal cell death induced by 300 microm KCN from 83.6 to 43.5%. However, at 1-100 mm KCN, the protective effects of LED decreased, and neuronal deaths increased. LED significantly restored neuronal ATP content only at 10 microm KCN but not at higher concentrations of KCN tested. Pretreatment with LED enhanced efficacy of LED during exposure to 10 or 100 microm KCN but did not restore enzyme activity to control levels. In contrast, LED was able to completely reverse the detrimental effect of tetrodotoxin, which only indirectly down-regulated enzyme levels. Among the wavelengths tested (670, 728, 770, 830, and 880 nm), the most effective ones (830 nm, 670 nm) paralleled the NIR absorption spectrum of oxidized cytochrome c oxidase, whereas the least effective wavelength, 728 nm, did not. The results are consistent with our hypothesis that the mechanism of photobiomodulation involves the up-regulation of cytochrome c oxidase, leading to increased energy metabolism in neurons functionally inactivated by toxins.     

Disruption of the coenzyme binding site and dimer interface revealed in the crystal structure of mitochondrial aldehyde dehydrogenase "Asian" variant

Mitochondrial aldehyde dehydrogenase (ALDH2) is the major enzyme that oxidizes ethanol-derived acetaldehyde. A nearly inactive form of the enzyme, ALDH2*2, is found in about 40% of the East Asian population. This variant enzyme is defined by a glutamate to lysine substitution at residue 487 located within the oligomerization domain. ALDH2*2 has an increased Km for its coenzyme, NAD+, and a decreased kcat, which lead to low activity in vivo. Here we report the 2.1 A crystal structure of ALDH2*2. The structure shows a large disordered region located at the dimer interface that includes much of the coenzyme binding cleft and a loop of residues that form the base of the active site. As a consequence of these structural changes, the variant enzyme exhibits rigid body rotations of its catalytic and coenzyme-binding domains relative to the oligomerization domain. These structural perturbations are the direct result of the inability of lysine 487 to form important stabilizing hydrogen bonds with arginines 264 and 475. Thus, the elevated Km for coenzyme exhibited by this variant probably reflects the energetic penalty for reestablishing this site for productive coenzyme binding, whereas the structural alterations near the active site are consistent with the lowered Vmax.     

Reverse engineering the L-type Ca2+ channel alpha1c subunit in adult cardiac myocytes using novel adenoviral vectors

The alpha(1c) subunit of the cardiac L-type Ca(2+) channel, which contains the channel pore, voltage- and Ca(2+)-dependent gating structures, and drug binding sites, has been well studied in heterologous expression systems, but many aspects of L-type Ca(2+) channel behavior in intact cardiomyocytes remain poorly characterized. Here, we develop adenoviral constructs with E1, E3 and fiber gene deletions, to allow incorporation of full-length alpha(1c) gene cassettes into the adenovirus backbone. Wild-type (alpha(1c-wt)) and mutant (alpha(1c-D-)) Ca(2+) channel adenoviruses were constructed. The alpha(1c-D-) contained four point substitutions at amino acid residues known to be critical for dihydropyridine binding. Both alpha(1c-wt) and alpha(1c-D-) expressed robustly in A549 cells (peak L-type Ca(2+) current (I(CaL)) at 0 mV: alpha(1c-wt) -9.94+/-1.00pA/pF, n=9; alpha(1c-D-) -10.30pA/pF, n=12). I(CaL) carried by alpha(1c-D-) was markedly less sensitive to nitrendipine (IC(50) 17.1 microM) than alpha(1c-wt) (IC(50) 88 nM); a feature exploited to discriminate between engineered and native currents in transduced guinea-pig myocytes. 10 microM nitrendipine blocked only 51+/-5% (n=9) of I(CaL) in alpha(1c-D-)-expressing myocytes, in comparison to 86+/-8% (n=9) of I(CaL) in control myocytes. Moreover, in 20 microM nitrendipine, calcium transients could still be evoked in alpha(1c-D-)-transduced cells, but were largely blocked in control myocytes, indicating that the engineered channels were coupled to sarcoplasmic reticular Ca(2+) release. These alpha(1c) adenoviruses provide an unprecedented tool for structure-function studies of cardiac excitation-contraction coupling and L-type Ca(2+) channel regulation in the native myocyte background.     

Old and new data, new issues: the mitochondrial DeltaPsi

New and old data pertinent to the electrochemical potentials across the inner mitochondrial membrane are reviewed with the intent of reconciling the various findings in the light of new perspectives provided by more recent knowledge. A careful scrutiny of old data permits ruling out the presence of a significant metabolically dependent electrical membrane potential. Recent technological advances make it possible to test the proposed alternatives. These proposals recast the original idea, and the possible mechanisms that are emerging also invoke a protonmotive force. Our conclusions that DeltaPsi is not involved in oxidative-phosphorylation finds parallel observations in Halobacterium halobium [H. Michel, D. Oesterhelt, Electrochemical proton gradient across the cell membrane of Halobacterium halobium: comparison of the light-induced increase with the increase of intracellular adenosine triphosphate under steady-state illumination, Biochemistry 19 (1980) 4615-4619] and thylakoid vesicles [D.R. Ort, R.A. Dilley, N.E. Good, Photophosphorylation as a function of illumination time II. Effects of permeant buffers, Biochim. Biophys. Acta 449 (1976) 108-129] in which light-induced ATP synthesis occurs in the absence of an apparent DeltaPsi or DeltapH, suggesting the presence of mechanisms similar to the one proposed for mitochondria.