A new bioassay identifies proliferation ratios of fibroblasts and myofibroblasts.

Myofibroblasts are resident cells of wound healing, contractures and fibroses; these tissues are often referred to as fibroproliferative. Whether myofibroblasts themselves proliferate is of interest. Since many in vitro cultures are heterogeneous, staining in situ is required to identify the myofibroblast. We have tested a newly available fluorescent staining kit using ethynyl deoxyuridine (EdU) and click chemistry to identify EdU incorporation into the replicated DNA of proliferative cells. The proliferation stain was combined with the definitive myofibroblast immunostain for alpha smooth muscle actin (α-sma). Fibroblasts were grown on coverslips and within attached collagen lattices. Cultures were pulsed with EdU 4 h prior to fixation. Different standard methods of fixation and permeabilization were used to test the effects of these variables on EdU and α-sma labeling. Images of the stained samples were quantified as the total percentage of proliferative cells, as well as the proportion of fibroblasts and myofibroblasts that were proliferating. Proliferative myofibroblasts were identified in both culture conditions and with all preparation methods tested. Proliferation within the fibroblast population was greater than within the myofibroblast population in both culture conditions. Fixation and permeabilization had little effect on EdU or α-sma labeling. This method of identifying proliferative myofibroblasts will be useful in future studies of myofibroblast proliferation within heterogeneous populations.

Distance-dependent, pair potential for protein folding: results from linear optimization.

The results of an optimization of a folding potential are reported. The complete energy function is modeled as a sum of pairwise interactions with a flexible functional form. The relevant distance between two amino acids (2 - 9 A) is divided into 13 intervals, and the energy of each interval is optimized independently. We show, in accord with a previous publication (Tobi et al., Proteins 2000;40:71-85) that it is impossible to find a pair potential with the above flexible form that recognizes all native folds. Nevertheless, a potential that rates correctly a subset of the decoy structures was constructed and optimized. The resulting potential is compared with a distance-dependent statistical potential of Bahar and Jernigan. It is further tested against decoy structures that were created in the Levitt's group. On average, the new potential places native shapes lower in energy and provides higher Z scores than other potentials.

On the design and analysis of protein folding potentials.

Pairwise interaction models to recognize native folds are designed and analyzed. Different sets of parameters are considered but the focus was on 20 x 20 contact matrices. Simultaneous solution of inequalities and minimization of the variance of the energy find matrices that recognize exactly the native folds of 572 sequences and structures from the protein data bank (PDB). The set includes many homologous pairs, which present a difficult recognition problem. Significant recognition ability is recovered with a small number of parameters (e.g., the H/P model). However, full recognition requires a complete set of amino acids. In addition to structures from the PDB, a folding program (MONSSTER) was used to generate decoy structures for 75 proteins. It is impossible to recognize all the native structures of the extended set by contact potentials. We therefore searched for a new functional form. An energy function U, which is based on a sum of general pairwise interactions limited to a resolution of 1 angstrom, is considered. This set was infeasible too. We therefore conjecture that it is not possible to find a folding potential, resolved to 1 angstrom, which is a sum of pair interactions.

Coupling the folding of homologous proteins.

The empirical observation that homologous proteins fold to similar structures is used to enhance the capabilities of an ab initio algorithm to predict protein conformations. A penalty function that forces homologous proteins to look alike is added to the potential and is employed in the coupled energy optimization of several homologous proteins. Significant improvement in the quality of the computed structures (as compared with the computational folding of a single protein) is demonstrated and discussed.

N-type voltage-sensitive calcium channel interacts with syntaxin, synaptotagmin and SNAP-25 in a multiprotein complex.

Expression of the N-type voltage sensitive calcium channel in Xenopus oocytes along with syntaxin and p65 showed that the syntaxin-modified N-type channel properties, were fully reversed by p65. The inward current was restored to a significantly higher amplitude when all three proteins were present, suggesting that the channel interacts with syntaxin, p65 and SNAP-25 in a quaternary complex. Further support to a multicomplex formation between the channel and the synaptic proteins was drawn from the steady-state voltage inactivation profiles. A physical interaction of the N-type calcium channel with the vesicular protein synaptotagmin (p65) was demonstrated biochemically, using recombinant fusion proteins. The interaction is confined to a cytosolic channel domain that separates segments II and III amino acids 710-1090 of the N-type channel (N-Loop710-1090). In vitro binding of recombinant N-Loop710-1090 to p65 (amino acids 96-421) involves the two C2 domains of p65, C2A domain [amino acids 96-265; p65(1-3)] and C2B domain [amino acids 248-421; p65(3-5)]. While the binding of C2A and C2B domains was calcium independent, C2B domain binding to the N-Loop was inositol-hexaphosphate (IP6)-sensitive. The N-Loop710-1090 binding to p65 was competed by syntaxin and SNAP-25, which are synaptic plasma membrane proteins. These combined functional and biochemical approaches provide evidence for a complex formation between the N-type channel and the exocytotic machinery which by generating fusion-competent vesicles may function to regulate the process of synaptic secretion.

Synaptotagmin restores kinetic properties of a syntaxin-associated N-type voltage sensitive calcium channel.

The voltage sensitive N-type calcium channel interacts functionally and biochemically with synaptotagmin (p65). N-type channel interaction with p65 is demonstrated in the Xenopus oocyte expression system, where p65 alters the steady state voltage inactivation of the N-channel, and fully restores the syntaxin-modified current amplitude and inactivation kinetics in a calcium dependent manner. In agreement with the functional results, GST-p65 fusion protein binds to a cytosolic region, amino acids 710-1090 of the N-type channel (N-loop(710-1090)). The results of the combined approach provide a functional and biochemical basis for proposing that p65 interaction with the N-type channel brings p65 into a close association with a syntaxin-coupled channel. In turn, calcium entry through the liberated channel initiates fusion of the primed vesicles with the cell membrane at a short distance from the site of calcium entry.

The alpha 2/delta subunit of voltage sensitive Ca2+ channels is a single transmembrane extracellular protein which is involved in regulated secretion.

The membrane topology of alpha 2/delta subunit was investigated utilizing electrophysiological functional assay and specific anti-alpha 2 antibodies. (a) cRNA encoding a deleted alpha 2/delta subunit was coinjected with alpha 1C subunit of the L-type calcium channel into Xenopus oocytes. The truncated form, lacking the third putative TM domain (alpha 2/delta delta TMIII), failed to amplify the expressed inward currents, normally induced by alpha 1C coinjected with intact alpha 2/delta subunit. Western blot analysis of alpha 2/delta delta TMIII shows the appearance of a degraded alpha 2 protein and no expression of the full-size two-TM truncated-protein. The improper processing of alpha 2/delta delta TMIII suggests that the alpha 2/delta is a single TM domain protein and the TM region is positioned at the delta subunit. (b) External application of anti-alpha 2 antibodies, prepared for an epitope within the alternatively spliced and 'intracellular' region, inhibits depolarization induced secretion in PC12, further supporting an external location of the alpha 2 subunit and establishing delta subunit as the only membrane anchor for the extracellular alpha 2 subunit.