[Expression of HCN4 protein in ventricular outflow tract of rabbit with idiopathic ventricular tachycardia].

OBJECTIVE: To confirm the existence of purkinje fibers in rabbit outflow tract tissue and explore the role of Hyperpolarization-Activated Cyclic Nucleotide-Gated Channel 4 (HCN4) protein in idiopathic ventricular tachycardia. METHODS: A total of ten New Zealand white rabbits were randomly selected to observe whether there were pukinje fibers in outflow tract by the methods of HE staining and immunohistochemical detection of midsize neurofilament (NF-M). Forty rabbits were randomly divided into four groups: normal control group (SO), ventricular tachycardia group (VT), ventricular tachycardia+ esmolol intervention group (VT+ ESM) and ventricular tachycardia+ ivabradine intervention group (VT+ IVA). Immunohistochemistry was used to detect the expression of HCN4 protein in ventricular outflow tract; the required output voltage amplitude was recorded when ventricular tachycardia was induced; the times and duration of spontaneous ventricular tachycardia when stimulation stopped were also recorded for each group. RESULTS: (1)Purkinje fibers existed in the myocardial tissue of rabbit outflow tract. (2)HCN4 protein expression significantly increased in VT group compared with SO group (left ventricular: 97.6 ± 16.7 vs 29.0 ± 8.0, P<0.01; right ventricular: 92.7 ± 12.3 vs 26.0 ± 10.8, P<0.01), the expression of HCN4 protein obviously reduced in VT+ IVA group compared with VT group (left ventricular: 32.0 ± 9.4 vs 97.6 ± 16.7, P<0.01; right ventricular: 30.8 ± 12.4 vs 92.7 ± 12.3, P<0.01). (3)The output voltage amplitude required to induce the desired ventricular tachycardia in VT+ ESM group and VT+ IVA group were higher than the VT group, under the same high frequency stimulation (P<0.01); the times and duration of spontaneous ventricular tachycardia in VT+ ESM group and VT+ IVA group significantly reduced when the stimulation stopped, compared with the VT group (both P<0.01). CONCLUSIONS: (1)Purkinje fibers exist in ventricular outflow tract, which may be the histological origin of the ventricular tachycardia. (2)HCN4 protein is up-regulated in ventricular outflow tract when ventricular tachycardia occurs. (3)Esmolol and ivabradine can prevent and reduce the occurrence of ventricular tachycardia, and as the specific inhibitor of the HCN channel, the effect of ivabradine is more obvious.

Effects of solutes on empirical phase diagrams of human fibroblast growth factor 1.

A variety of solutes are commonly used to increase the stability of protein in therapeutic formulations. An empirical phase diagram approach is used to evaluate the effects of different types of additives on the solution behavior of a protein of pharmaceutical interest, human fibroblast growth factor 1 (FGF-1). A specific stabilizer, heparin, and a nonspecific stabilizer, sucrose, were used in this work. The protein was characterized as a function of pH (3-8) and temperature (10-85 degrees C) using Far-UV circular dichroism (Far-UV CD), intrinsic and extrinsic fluorescence as well as second derivative UV absorption spectroscopy. Empirical phase diagrams were constructed to summarize the biophysical characterization data obtained with FGF-1 alone, in the presence of a threefold weight excess of heparin (3x heparin) or 10% sucrose (w/v). Three phases are observed in the low temperature regions at pH 3, 4, and 5-8. Phase boundaries corresponding to major heat-induced transitions are detected in the physiological temperature range. The highest thermal stabilities are observed near neutral pH (pH 6 and 7). Both heparin and sucrose appear to enhance the thermal stability of FGF-1, although their effects on the phase diagram are quite distinct. The greatest stabilization is observed at pH 8. Only heparin appears to protect FGF-1 from acid-induced unfolding to any extent.

Characterization of TAT-mediated transport of detachable kinase substrates.

The conjugation of peptides derived from the HIV TAT protein to membrane-impermeant molecules has gained wide acceptance as a means for intracellular delivery. Numerous studies have addressed the mechanism of uptake and kinetics of TAT translocation, but the cytosolic concentrations and bioavailability of the transported cargo have not been well-characterized. The current paper utilizes a microanalytical assay to perform quantitative single-cell measurements of the concentration and accessibility of peptide-based substrates for protein kinase B (PKB) and Ca(2+)/calmodulin-activated kinase II. The substrate peptide and TAT were conjugated through a releasable linker, either a disulfide or photolabile bond. Free substrate peptide concentrations of approximately 10(-20)-10(-18) moles were attainable in a cell when substrates were delivered utilizing these conjugates. The substrate peptides delivered as a disulfide conjugate were often present in the cytosol as several oxidized forms. Brief exposure of cells loaded with the photolabile conjugates to UVA light released free substrate peptide into the cytosol. Substrate peptide delivered by either conjugate was accessible to cytosolic kinase as demonstrated by the efficient phosphorylation of the peptide when the appropriate kinase was active. After incubation of the conjugated substrate with cells, free, kinase-accessible substrate was detectable in less than 30 min. Release of the majority of loaded substrate peptide from sequestered organelles occurred within 1 h. The utility of the photocleavable conjugates was demonstrated by measuring the activation of PKB in 3T3 cells after addition of varying concentrations of platelet-derived growth factor.

A quantitative single-cell assay for protein kinase B reveals important insights into the biochemical behavior of an intracellular substrate peptide.

The introduction of peptides into living cells for the purpose of manipulating cellular biochemistry has become widespread throughout biology. However, little is known about the behavior of these short sequences of amino acids within cells, particularly those used as substrates or inhibitors for kinases and other enzymes. We utilized a quantitative, single-cell assay to demonstrate that an 11-amino acid peptide was efficiently phosphorylated by intracellular protein kinase B (PKB) in fibrosarcoma cell line HT1080 and in NIH-3T3 cells. The phosphorylated peptide was also readily dephosphorylated by intracellular phosphatases. Assays of the peptide's phosphorylation in single, living cells measured the balance of the activities of PKB and phosphatases in that cell. At a peptide concentration below the K(M) of PKB and the phosphatases, the ratio of phosphorylated to nonphosphorylated peptide at the steady state was independent of the peptide concentration. A single-cell assay utilizing this peptide revealed the existence of two subpopulations of cells whose unique activities had hitherto been obscured by population averaging. Additional studies of cells stimulated by PDGF demonstrated that a quantitative analysis of PKB activation in response to a physiological stimulus was possible. These studies demonstrated that short peptides can remain specific within the complex intracellular milieu and function as sensitive reporters of the activation state of native kinases within live cells.

Separation of mixtures of acidic and basic peptides at neutral pH.

Mixtures of acidic and basic peptides composed of the phosphorylated and nonphosphorylated forms of peptide substrates for kinases and a phosphatase were separated by capillary electrophoresis (CE) in buffer conditions compatible with live mammalian cells. The separation of such mixtures was especially challenging given the high salt and neutral pH of the requisite physiologic buffers. Due to poor peak reproducibility in bare capillaries, several strategies were implemented to improve the electrophoretic separation of the peptide mixtures. Covalent coating of the capillary with the neutral polymer poly(dimethylacrylamide) (PDMA) resulted in a 2-fold improvement in the migration time RSD, but required the use of hydrodynamic flow to overcome the differing electrophoretic mobilities (microeo) of the peptides at neutral pH. This parabolic fluid flow diminished separation efficiency almost 5-fold. Polarity switching during the CE run was used to overcome the opposed microeo, but required the retention of hydrodynamic flow and consequent reduction in separation efficiency. The most efficient separations were seen with the use of covalently-linked, charged polymer coatings to maintain electroosmotic flow and to reduce wall interactions. Two such coatings were tested in the current study. Relative to the PDMA coating, an anionic poly(acrylate) improved the average migration time RSD of six peptides from 1.3 to 0.85% and average separation efficiency from 4.8 to 18.0 (x 10(4) plates/m). Likewise, cationic poly([3-(methacryloylamino)propyl]-trimethylammonium) improved the average migration time RSD of eight peptides from 1.2 to 1.1% and average separation efficiency from 4.8 to 33.9 (x 10(4) plates/m). These findings will be of value to the growing number of applications for analytical techniques utilizing CE for cellular analysis and biochemical studies.