Large-conductance calcium-activated potassium channel activity is absent in human and mouse neutrophils and is not required for innate immunity

Large-conductance Ca²⁺-activated K⁺ (BK) channels are reported to be essential for NADPH oxidase-dependent microbial killing and innate immunity in leukocytes. Using human peripheral blood and mouse bone marrow neutrophils, pharmacological targeting, and BK channel gene-deficient (BK^–/–) mice, we stimulated NADPH oxidase activity with 12-O-tetradecanoylphorbol-13-acetate (PMA) and performed patch-clamp recordings on isolated neutrophils. Although PMA stimulated NADPH oxidase activity as assessed by O₂^– and H₂O₂ production, our patch-clamp experiments failed to show PMA-activated BK channel currents in neutrophils. In our studies, PMA induced slowly activating currents, which were insensitive to the BK channel inhibitor iberiotoxin. Instead, the currents were blocked by Zn²⁺, which indicates activation of proton channel currents. BK channels are gated by elevated intracellular Ca²⁺ and membrane depolarization. We did not observe BK channel currents, even during extreme depolarization to +140 mV and after elevation of intracellular Ca²⁺ by N-formyl-L-methionyl-L-leucyl-phenylalanine. As a control, we examined BK channel currents in cerebral and tibial artery smooth muscle cells, which showed characteristic BK channel current pharmacology. Iberiotoxin did not block killing of Staphylococcus aureus or Candida albicans. Moreover, we addressed the role of BK channels in a systemic S. aureus and Yersinia enterocolitica mouse infection model. After 3 and 5 days of infection, we found no differences in the number of bacteria in spleen and kidney between BK^–/– and BK^+/+ mice. In conclusion, our experiments failed to identify functional BK channels in neutrophils. We therefore conclude that BK channels are not essential for innate immunity. killing assay; reactive oxygen species; BK-deficient mice; mice infection     

Der1-mediated preprotein import into the periplastid compartment of chromalveolates?

Phototrophic chromalveolates possess plastids surrounded by either 3 or 4 membranes, revealing their secondary endosymbiotic origin from an engulfed eukaryotic alga. In cryptophytes, a member of the chromalveolates, the organelle is embedded within a designated region of the host's rough endoplasmic reticulum (RER). Its eukaryotic compartments other than the plastid were reduced to the mere remains of its former cytosol, the periplastid compartment (PPC, PP space), and its nucleus, the nucleomorph, separated from the RER by its former plasma membrane, the periplast membrane (PPM). In the nucleomorph genome of the cryptophyte Guillardia theta, we identified several genes sharing homology with components of the ER-associated degradation (ERAD) machinery of yeast and higher eukaryotes, namely ORF201 and ORF477, homologs of membrane-bound proteins, Der1p (Degradation in the ER protein 1) and the RING-finger ubiquitin ligase Hrd1, and a truncated version of Udf1, a cofactor of Cdc48, a lumenal ATPase. Exemplarily, studies on the Der1-homolog ORF201 showed that this protein partially rescued a yeast deletion mutant, indicating the existence of a functional PPC-specific ERAD-like system in cryptophytes. With the noninvestigated exception of haptophytes a phylogenetically and mechanistically related system is apparently present in all chromalveolates with 4 membrane-bound plastids because amongst others, PPC-specific Derlins (Der1-like proteins), CDC48 and its cofactor Ufd1 were identified in the nuclear genomes of diatoms and apicomplexa. These proteins are equipped with the required topogenic signals to direct them into the periplastid compartment of their secondary symbionts. Based on our findings, we suggest that all chromalveolates with 4 membrane-bound plastids express an ERAD-derived machinery in the PPM of their secondary plastid, coexisting physically and systematically adjacent to the host's own ERAD system. We propose herewith that this system was functionally adapted to mediate transport of nucleus-encoded PPC/plastid preproteins from the RER into the periplastid space.     

The transition zone protein Rpgrip1l regulates proteasomal activity at the primary cilium

Mutations in RPGRIP1L result in severe human diseases called ciliopathies. To unravel the molecular function of RPGRIP1L, we analyzed Rpgrip1l^−/− mouse embryos, which display a ciliopathy phenotype and die, at the latest, around birth. In these embryos, cilia-mediated signaling was severely disturbed. Defects in Shh signaling suggested that the Rpgrip1l deficiency causes an impairment of protein degradation and protein processing. Indeed, we detected a cilia-dependent decreased proteasomal activity in the absence of Rpgrip1l. We found different proteasomal components localized to cilia and identified Psmd2, a component of the regulatory proteasomal 19S subunit, as an interaction partner for Rpgrip1l. Quantifications of proteasomal substrates demonstrated that Rpgrip1l regulates proteasomal activity specifically at the basal body. Our study suggests that Rpgrip1l controls ciliary signaling by regulating the activity of the ciliary proteasome via Psmd2.     

Automatic Scoring and Quality Assessment Using Accuracy Bounds for FP-TDI SNP Genotyping Data

BACKGROUND: Human diversity, namely single nucleotide polymorphisms (SNPs), is becoming a focus of biomedical research. Despite the binary nature of SNP determination, the majority of genotyping assay data need a critical evaluation for genotype calling. We applied statistical models to improve the automated analysis of 2-dimensional SNP data. METHODS: We derived several quantities in the framework of Gaussian mixture models that provide figures of merit to objectively measure the data quality. The accuracy of individual observations is scored as the probability of belonging to a certain genotype cluster, while the assay quality is measured by the overlap between the genotype clusters. RESULTS: The approach was extensively tested with a dataset of 438 nonredundant SNP assays comprising >150,000 datapoints. The performance of our automatic scoring method was compared with manual assignments. The agreement for the overall assay quality is remarkably good, and individual observations were scored differently by man and machine in 2.6% of cases, when applying stringent probability threshold values. CONCLUSION: Our definition of bounds for the accuracy for complete assays in terms of misclassification probabilities goes beyond other proposed analysis methods. We expect the scoring method to minimise human intervention and provide a more objective error estimate in genotype calling.     

CD40 ligation prevents onset of tolerogenic properties in human dendritic cells treated with CTLA-4-Ig

The synthetic immunomodulator cytotoxic T lymphocyte antigen 4-Ig (CTLA-4-Ig) initiates effects in human monocyte-derived dendritic cells (DC) that rely on immunosuppressive tryptophan catabolism. However, it is unable to induce suppressive properties in DC matured by CD40 engagement. Thus, CD40-driven events may physiologically set human DC free from restraint by regulatory cells expressing surface CTLA-4.