Engineering of a novel Ca²⁺-regulated kinesin molecular motor using a calmodulin dimer linker

The kinesin-microtubule system holds great promise as a molecular shuttle device within biochips. However, one current barrier is that such shuttles do not have "on-off" control of their movement. Here we report the development of a novel molecular motor powered by an accelerator and brake system, using a kinesin monomer and a calmodulin (CaM) dimer. The kinesin monomer, K355, was fused with a CaM target peptide (M13 peptide) at the C-terminal part of the neck region (K355-M13). We also prepared CaM dimers using CaM mutants (Q3C), (R86C), or (A147C) and crosslinkers that react with cysteine residues. Following induction of K355-M13 dimerization with CaM dimers, we measured K355-M13 motility and found that it can be reversibly regulated in a Ca(2+)-dependent manner. We also found that velocities of K355-M13 varied depending on the type and crosslink position of the CaM dimer used; crosslink length also had a moderate effect on motility. These results suggest Ca(2+)-dependent dimerization of K355-M13 could be used as a novel molecular shuttle, equipped with an accelerator and brake system, for biochip applications.     

The roles of amino acid residues at positions 216 and 219 in the structural stability and metabolic functions of rat cytochrome P450 2D1 and 2D2

We examined the effects of the mutual substitution of amino acid residues at positions 216 and 219 between rat CYP2D1 and CYP2D2 on their microsomal contents and enzymatic functions using a yeast cell expression system and 5-methoxy-N,N-diisopropyltryptamine (5-MeO-DIPT) as a substrate. CYP2D1 has amino acid residues, leucine and valine, at positions of 216 and 219, respectively, whereas CYP2D2 has phenylalanine and aspartic acid at the same positions. In reduced carbon monoxide-difference spectroscopic analysis, the substitution of Asp-219 of CYP2D2 by valine markedly increased a peak at 450 nm and concomitantly decreased a peak at 420 nm, while the replacement of Phe-216 of CYP2D2 with leucine gave no observable change. The double substitution of Phe-216 and Asp-219 by leucine and valine, respectively, yielded a typical CYP spectrum. The substitution of Val-219 of CYP2D1 by aspartic acid decreased the CYP content to one-half, whereas the replacement of Leu-216 with phenylalanine did not have any effect. The double substitution of Leu-216 and Val-219 of CYP2D1 by phenylalanine and aspartic acid, respectively, diminished the CYP content by 90%. CYP2D1 catalyzed both 5-MeO-DIPT N-deisopropylation and O-demethylation at relatively low levels, while CYP2D2 catalyzed 5-MeO-DIPT O-demethylation efficiently. The substitution of the amino acid at position 216 substantially increased 5-MeO-DIPT oxidation activities of the two CYP2D enzymes. The replacement of the amino acid at position 219 increased the 5-MeO-DIPT O- and N-dealkylation activities of CYP2D1, whereas it decreased the 5-MeO-DIPT O-demethylation activity of CYP2D2. These results indicate that amino acid residues at positions 216 and 219 have important roles in the enzymatic functions of rat CYP2D1 and CYP2D2.     

Regulation of the paracellular Na+ and Cl- conductances by the NaCl-generated osmotic gradient in a manner dependent on the direction of osmotic gradients

In the present study, we investigated the effect of osmolality on the paracellular ion conductance (Gp) composed of the Na⁺ conductance (G_Na) and the Cl⁻ conductance (G_Cl). An osmotic gradient generated by NaCl with relatively apical hypertonicity (NaCl-absorption-direction) induced a large increase in the G_Na associated with a small increase in the G_Cl, whereas an osmotic gradient generated by NaCl with relatively basolateral hypertonicity (NaCl-secretion-direction) induced small increases in the G_Na and the G_Cl. These increases in the Gp caused by NaCl-generated osmotic gradients were diminished by the application of sucrose canceling the NaCl-generated osmotic gradient. The osmotic gradient generated by basolateral application of sucrose without any NaCl gradients had little effects on the Gp. However, this basolateral application of sucrose produced a precondition drastically quickening the time course of the action of the NaCl-generated osmotic gradient on the Gp. Further, we found that application of the basolateral hypotonicity generated by reduction of NaCl concentration shifted the localization of claudin-1 to the apical from the basolateral side. These results indicate that the osmotic gradient regulates the paracellular ion conductive pathway of tight junctions via a mechanism dependent on the direction of NaCl gradients associated with a shift of claudin-1 localization to the apical side in renal A6 epithelial cells.     

Determination of Reduced, Protein-Unbound, and Total Concentrations of N-Acetyl- -cysteine and -Cysteine in Rat Plasma by Postcolumn Ligand Substitution High-Performance Liquid Chromatography

A high-performance liquid chromatographic assay was developed for the quantitative determination of the sulfur-containing amino acids N-acetyl- -cysteine (NAC) and -cysteine (Cys) in rat plasma. The thiols were separated by reverse-phase ion-pair chromatography, and the column eluent was continuously mixed with an iodoplatinate-containing solution. The substitution of sulfur of the thiol compound with iodide was quantitatively determined by measuring changes in the absorption at 500 nm. The low-molecular-weight disulfides and mixed disulfide conjugates of thiols with proteins were entirely reduced to the original reduced compounds by dithiothreitol. By reducing these two types of disulfides separately during sample pretreatment, the reduced, protein-unbound, and total thiol concentrations could also be determined. Validation testing was performed, and no problems were encountered. The limit of detection was approximately 20 pmol of thiol on the column. The present method was used to measure the plasma concentrations of NAC and Cys in the rat after a bolus intravenous administration of NAC, focusing on disulfide formation. The binding of NAC to protein through mixed disulfide formation proceeds in a time-dependent and reversible manner. Moreover, this “stable” covalent binding might limit total drug elimination, while the unbound NAC is rapidly eliminated. Consequently, the analytical method described in this study is very useful for the determination of plasma NAC and Cys, including disulfide conjugates derived from them.     

Role of amino acid residue 90 in bioactivity and receptor binding capacity of tumor necrosis factor mutants

We have previously produced two bioactive lysine-deficient mutants of TNF-alpha (mutTNF-K90R,-K90P) and found that these mutants have bioactivity superior to wild-type TNF (wtTNF). Because these mutants contained same amino acid except for amino acid 90, it is unclear which amino acid residue is optimal for showing bioactivity. We speculated that this amino acid position was exchangeable, and this amino acid substitution enabled the creation of lysine-deficient mutants with enhanced bioactivity. Therefore, we produced mutTNF-K90R variants (mutTNF-R90X), in which R90 was replaced with other amino acids, to assay their bioactivities and investigated the importance of amino acid position 90. As a result, mutTNF-R90X that replaced R90 with lysine, arginine and proline were bioactive, while other mutants were not bioactive. Moreover, these three mutants showed bioactivity as good as or better than wtTNF. R90 replaced with lysine or arginine had especially superior binding affinities. These results suggest that the amino acid position 90 in TNF-alpha is important for TNF-alpha bioactivity and could be altered to improve its bioactivity to generate a "super-agonist".     

NF-kappaB mediates the survival of human bronchial epithelial cells exposed to cigarette smoke extract

Background

We have previously reported that low concentrations of cigarette smoke extract induce DNA damage without leading to apoptosis or necrosis in human bronchial epithelial cells (HBECs), and that IL-6/STAT3 signaling contributes to the cell survival. Since NF-κB is also involved in regulating apoptosis and cell survival, the current study was designed to investigate the role of NF-κB in mediating cell survival in response to cigarette smoke exposure in HBECs.

Methods

Both the pharmacologic inhibitor of NF-κB, curcumin, and RNA interference targeting p65 were used to block NF-κB signaling in HBECs. Apoptosis and cell survival were then assessed by various methods including COMET assay, LIVE/DEAD Cytotoxicity/Viability assay and colony formation assay.

Results

Cigarette smoke extract (CSE) caused DNA damage and cell cycle arrest in S phase without leading to apoptosis in HBECs as evidenced by TUNEL assay, COMET assay and DNA content assay. CSE stimulated NF-κB -DNA binding activity and up-regulated Bcl-XL protein in HBECs. Inhibition of NF-κB by the pharmacologic inhibitor curcumin (20 μM) or suppression of p65 by siRNA resulted in a significant increase in cell death in response to cigarette smoke exposure. Furthermore, cells lacking p65 were incapable of forming cellular colonies when these cells were exposed to CSE, while they behaved normally in the regular culture medium.

Conclusion

The current study demonstrates that CSE activates NF-κB and up-regulates Bcl-XL through NF-kB activation in HBECs, and that CSE induces cell death in cells lacking p65. These results suggest that activation of NF-κB regulates cell survival following DNA damage by cigarette smoke in human bronchial epithelial cells.