Modulation of MICA on the surface of Chlamydia trachomatis-infected endocervical epithelial cells promotes NK cell-mediated killing

Chlamydia trachomatis serovars D-K are obligate intracellular bacteria that have tropism for the columnar epithelial cells of the genital tract. Chlamydia trachomatis infection has been reported to induce modifications in immune cell ligand expression on epithelial host cells. In this study, we used an in vitro infection model that resulted in a partial infection of C. trachomatis-exposed primary-like immortalized endocervical epithelial cells (A2EN). Using this model, we demonstrated that expression of the natural killer (NK) cell activating ligand, MHC class I-related protein A (MICA), was upregulated on C. trachomatis-infected, but not on noninfected bystander cells. MICA upregulation was concomitant with MHC class I downregulation and impacted the susceptibility of C. trachomatis-infected cells to NK cell activity. The specificity of MICA upregulation was reflected by a higher cytolytic activity of an NK cell line (NK92MI) against C. trachomatis-infected cells compared with uninfected control cells. Significantly, data also indicated that NK cells exerted a partial, but incomplete sterilizing effect on C. trachomatis as shown by the reduction in recoverable inclusion forming units (IFU) when cocultured with C. trachomatis-infected cells. Taken together, our data suggest that NK cells may play a significant role in the ability of the host to counter C. trachomatis infection.     

The dual-oscillator system of Drosophila melanogaster under natural-like temperature cycles

Dual-oscillator systems that control morning and evening activities can be found in a wide range of animals. The two coupled oscillators track dawn and dusk and flexibly adapt their phase relationship to seasonal changes. This is also true for the fruit fly Drosophila melanogaster that serves as model organism to understand the molecular and anatomical bases of the dual-oscillator system. In the present study, the authors investigated which temperature parameters are crucial for timing morning and evening activity peaks by applying natural-like temperature cycles with different daylengths. The authors found that the morning peak synchronizes to the temperature increase in the morning and the evening peak to the temperature decrease in the afternoon. The two peaks did not occur at fixed absolute temperatures, but responded flexibly to daylength and overall temperature level. Especially, the phase of the evening peak clearly depended on the absolute temperature level: it was delayed at high temperatures, whereas the phase of the M peak was less influenced. This suggests that the two oscillators have different temperature sensitivities. The bimodal activity rhythm was absent in the circadian clock mutants Clk(Jrk) and cyc(01) and reduced in per(01) and tim(01) mutants. Whereas the activity of Clk(Jrk) mutants just followed the temperature cycles, that of per(01) and tim(01) mutants did not, suggesting that these mutants are not completely clockless. This study revealed new characteristics of the dual-oscillator system in Drosophila that were not detected under different photoperiods.     

The ability of serum from alpha 1-antitrypsin-deficient patients to inhibit PRT-201, a recombinant human type I pancreatic elastase

PRT-201 is a recombinant human pancreatic elastase under development as a treatment for blood vessels to promote hemodialysis access patency. Proteases such as elastase are normally inactivated by antiproteases such as alpha 1-antitrypsin. It is unknown if serum from patients with alpha 1-antitrypsin deficiency will inhibit PRT-201 elastase activity. An assay for PRT-201 elastase activity in the presence of serum was developed and validated. PRT-201 elastase activity inhibition curves were developed using serum and also using purified alpha 1-antitrypsin and alpha 2-macroglobulin. Serum from 15 patients with documented alpha 1-antitrypsin deficiency, some of whom were receiving alpha 1-antitrypsin augmentation therapy, and four normal volunteers was analyzed. Serum from normal volunteers and patients with alpha 1-antitrypsin deficiency completely inactivated PRT-201 elastase activity in vitro. In the alpha 1-antitrypsin-deficient patients, the volume of serum necessary to inhibit elastase activity was related to the serum concentration of alpha 1-antitrypsin and augmentation therapy. Purified alpha 1-antitrypsin and alpha 2-macroglobulin were each alone capable of completely inhibiting PRT-201 elastase activity. It is unlikely that the use of PRT-201 will be associated with negative outcomes in patients with alpha 1-antitrypsin deficiency.     

Loss of thioredoxin function in retinas of mice overexpressing amyloid β

Amyloid β peptides (Aβ) have been implicated in the pathogenesis of age-related macular degeneration (ARMD) and glaucoma. In this study, retinas of mice overexpressing Aβ (Tg) were compared to those of wild-type mice (Wt) and analyzed for oxidative stress parameters. We observed a progressive decrease in all retinal cell layers, which was significantly greater in Tg mice at 14 months and culminated in loss of the outer retina at 18 months of age. We also observed higher levels of reactive oxygen species, glial fibrillary acidic protein, and hydroperoxide in Tg versus Wt mice (14 months). These effects were associated with phosphorylation/activation of the apoptosis signal kinase 1 and the p38 mitogen-activated kinase. Western blotting analysis revealed progressive increases in the levels of thioredoxin 1 and thioredoxin inhibitory protein in Tg compared to Wt mice. No changes were observed in the levels of thioredoxin reductase 1 (TrxR1); however, measurements of TrxR1 activity showed a 42.7±8% reduction in Tg mice versus Wt at 14 months of age. Our data suggest that Aβ-mediated retinal neurotoxicity involves impairment of the thioredoxin system and enhanced oxidative stress, potentially implicating this mechanism in the pathogenesis of ARMD and glaucoma.     

Phosphorylation of Ser-129 is the dominant pathological modification of alpha-synuclein in familial and sporadic Lewy body disease

A comprehensive, unbiased inventory of synuclein forms present in Lewy bodies from patients with dementia with Lewy bodies was carried out using two-dimensional immunoblot analysis, novel sandwich enzyme-linked immunosorbent assays with modification-specific synuclein antibodies, and mass spectroscopy. The predominant modification of alpha-synuclein in Lewy bodies is a single phosphorylation at Ser-129. In addition, there is a set of characteristic modifications that are present to a lesser extent, including ubiquitination at Lys residues 12, 21, and 23 and specific truncations at Asp-115, Asp-119, Asn-122, Tyr-133, and Asp-135. No other modifications are detectable by tandem mass spectrometry mapping, except for a ubiquitous N-terminal acetylation. Small amounts of Ser-129 phosphorylated and Asp-119-truncated alpha-synuclein are present in the soluble fraction of both normal and disease brains, suggesting that these Lewy body-associated forms are produced during normal metabolism of alpha-synuclein. In contrast, ubiquitination is only detected in Lewy bodies and is primarily present on phosphorylated synuclein; it therefore likely occurs after phosphorylated synuclein has deposited into Lewy bodies. This invariant pattern of specific phosphorylation, truncation, and ubiquitination is also present in the detergent-insoluble fraction of brain from patients with familial Parkinson's disease (synuclein A53T mutation) as well as multiple system atrophy, suggesting a common pathogenic pathway for both genetic and sporadic Lewy body diseases. These observations are most consistent with a model in which preferential accumulation of normally produced Ser-129 phosphorylated alpha-synuclein is the key event responsible for the formation of Lewy bodies in various Lewy body diseases.     

Ltv1 is required for efficient nuclear export of the ribosomal small subunit in Saccharomyces cerevisiae

In eukaryotes, 40S and 60S ribosomal subunits are assembled in the nucleus and exported to the cytoplasm independently of one another. Nuclear export of the 60S requires the adapter protein Nmd3, but no analogous adapter has been identified for the 40S. Ltv1 is a nonessential, nonribosomal protein that is required for 40S subunit biogenesis in yeast. Cells lacking LTV1 grow slowly, are hypersensitive to inhibitors of protein synthesis, and produce about half as many 40S subunits as do wild-type cells. Ltv1 interacts with Crm1, co-sediments in sucrose gradients with 43S/40S subunits, and copurifies with late 43S particles. Here we show that Ltv1 shuttles between nucleus and cytoplasm in a Crm1-dependent manner and that it contains a functional NES that is sufficient to direct the export of an NLS-containing reporter. Small subunit export is reduced in Deltaltv1 mutants, as judged by the altered distribution of the 5'-ITS1 rRNA and the 40S ribosomal protein RpS3. Finally, we show a genetic interaction between LTV1 and YRB2, a gene that encodes a Ran-GTP-, Crm1-binding protein that facilitates the small subunit export. We propose that Ltv1 functions as one of several possible adapter proteins that link the nuclear export machinery to the small subunit.     

The F-box protein Dia2 overcomes replication impedance to promote genome stability in Saccharomyces cerevisiae

The maintenance of DNA replication fork stability under conditions of DNA damage and at natural replication pause sites is essential for genome stability. Here, we describe a novel role for the F-box protein Dia2 in promoting genome stability in the budding yeast Saccharomyces cerevisiae. Like most other F-box proteins, Dia2 forms a Skp1-Cdc53/Cullin-F-box (SCF) E3 ubiquitin–ligase complex. Systematic analysis of genetic interactions between dia2Δ and ~4400 viable gene deletion mutants revealed synthetic lethal/synthetic sick interactions with a broad spectrum of DNA replication, recombination, checkpoint, and chromatin-remodeling pathways. dia2Δ strains exhibit constitutive activation of the checkpoint kinase Rad53 and elevated counts of endogenous DNA repair foci and are unable to overcome MMS-induced replicative stress. Notably, dia2Δ strains display a high rate of gross chromosomal rearrangements (GCRs) that involve the rDNA locus and an increase in extrachromosomal rDNA circle (ERC) formation, consistent with an observed enrichment of Dia2 in the nucleolus. These results suggest that Dia2 is essential for stable passage of replication forks through regions of damaged DNA and natural fragile regions, particularly the replication fork barrier (RFB) of rDNA repeat loci. We propose that the SCF^Dia2 ubiquitin ligase serves to modify or degrade protein substrates that would otherwise impede the replication fork in problematic regions of the genome.