Titanium Ions Promote Exogenous Calcium-Dependent Calcium Influx in Activated Jurkat T Cells: A Possible Mechanism to Explain Its Immunostimulatory Properties.

Titanium and its alloys have been widely used in dental and orthopedic implants. Owing to the biotribocorrosion behavior of implants in simulated oral environment, Ti(IV) ions could be released into surrounding tissues. Current studies have found that Ti(IV) ions could affect the biological activities of immune cells in adjacent tissues and subsequently jeopardize the long-term performance of implant prostheses. However, the potential mechanism underlying its immunomodulatory properties remains unclear. Calcium signaling has been confirmed to be involved in regulation of lymphocyte immune function. Therefore, we hypothesize that Ti(IV) ions modulated T cell function through the change of intracellular calcium concentrations. This study is aimed at exploring the role of intracellular calcium responses in the modulatory effect of Ti(IV) ions on unactivated and phytohemagglutinin-activated Jurkat T cells. Here, we confirmed that Ti(IV) ions within a certain concentration range induced CD69 expression on both unactivated and activated T cells in our study. Additionally, the combined stimulation with Ti(IV) ions and PHA increased expression of IL-1ß, TNF-α, and RANKL. Furthermore, we found that treatment with Ti(IV) induced a transitory increase in the levels of [Ca2+]i in activated Jurkat cells, dependent on the presence of exogenous calcium. Treatment with different doses of Ti(IV) for 24 h significantly increased the levels of [Ca2+]i in the activated Jurkat cells in a dose-dependent manner, but had little effect in the unactivated cells. Treatment with Ti(IV) did not significantly affect the PLCγ1 activation and inositol-1,4,5-trisphosphate (IP3) secretion in Jurkat cells. Taken together, these data indicated that Ti(IV) enhanced calcium influx during the T cell activation, independent of IP3-mediated intracellular calcium release. Our work provides insights into the mechanism involved in the regulation of lymphocyte behaviors under the effect of Ti(IV) ions, which may help to develop therapeutic strategies for dental implant failures.

Rates of gyrase supercoiling and transcription elongation control supercoil density in a bacterial chromosome.

Gyrase catalyzes negative supercoiling of DNA in an ATP-dependent reaction that helps condense bacterial chromosomes into a compact interwound "nucleoid." The supercoil density (σ) of prokaryotic DNA occurs in two forms. Diffusible supercoil density (σ(D)) moves freely around the chromosome in 10 kb domains, and constrained supercoil density (σ(C)) results from binding abundant proteins that bend, loop, or unwind DNA at many sites. Diffusible and constrained supercoils contribute roughly equally to the total in vivo negative supercoil density of WT cells, so σ = σ(C)+σ(D). Unexpectedly, Escherichia coli chromosomes have a 15% higher level of σ compared to Salmonella enterica. To decipher critical mechanisms that can change diffusible supercoil density of chromosomes, we analyzed strains of Salmonella using a 9 kb "supercoil sensor" inserted at ten positions around the genome. The sensor contains a complete Lac operon flanked by directly repeated resolvase binding sites, and the sensor can monitor both supercoil density and transcription elongation rates in WT and mutant strains. RNA transcription caused (-) supercoiling to increase upstream and decrease downstream of highly expressed genes. Excess upstream supercoiling was relaxed by Topo I, and gyrase replenished downstream supercoil losses to maintain an equilibrium state. Strains with TS gyrase mutations growing at permissive temperature exhibited significant supercoil losses varying from 30% of WT levels to a total loss of σ(D) at most chromosome locations. Supercoil losses were influenced by transcription because addition of rifampicin (Rif) caused supercoil density to rebound throughout the chromosome. Gyrase mutants that caused dramatic supercoil losses also reduced the transcription elongation rates throughout the genome. The observed link between RNA polymerase elongation speed and gyrase turnover suggests that bacteria with fast growth rates may generate higher supercoil densities than slow growing species.

A gyrase mutant with low activity disrupts supercoiling at the replication terminus.

When a mutation in an essential gene shows a temperature-sensitive phenotype, one usually assumes that the protein is inactive at nonpermissive temperature. DNA gyrase is an essential bacterial enzyme composed of two subunits, GyrA and GyrB. The gyrB652 mutation results from a single base change that substitutes a serine residue for arginine 436 (R436-S) in the GyrB protein. At 42 degrees C, strains with the gyrB652 allele stop DNA replication, and at 37 degrees C, such strains grow but have RecA-dependent SOS induction and show constitutive RecBCD-dependent DNA degradation. Surprisingly, the GyrB652 protein is not inactive at 42 degrees C in vivo or in vitro and it doesn't directly produce breaks in chromosomal DNA. Rather, this mutant has a low k(cat) compared to wild-type GyrB subunit. With more than twice the normal mean number of supercoil domains, this gyrase hypomorph is prone to fork collapse and topological chaos near the terminus of DNA replication.