Priming in the Type I-F CRISPR-Cas system triggers strand-independent spacer acquisition,bi-directionally from the primed protospacer

Clustered regularly interspaced short palindromic repeats (CRISPR), in combination with CRISPR associated (cas) genes, constitute CRISPR-Cas bacterial adaptive immune systems. To generate immunity, these systems acquire short sequences of nucleic acids from foreign invaders and incorporate these into their CRISPR arrays as spacers. This adaptation process is the least characterized step in CRISPR-Cas immunity. Here, we used Pectobacterium atrosepticum to investigate adaptation in Type I-F CRISPR-Cas systems. Pre-existing spacers that matched plasmids stimulated hyperactive primed acquisition and resulted in the incorporation of up to nine new spacers across all three native CRISPR arrays. Endogenous expression of the cas genes was sufficient, yet required, for priming. The new spacers inhibited conjugation and transformation, and interference was enhanced with increasing numbers of new spacers. We analyzed ∼350 new spacers acquired in priming events and identified a 5′-protospacer-GG-3′ protospacer adjacent motif. In contrast to priming in Type I-E systems, new spacers matched either plasmid strand and a biased distribution, including clustering near the primed protospacer, suggested a bi-directional translocation model for the Cas1:Cas2–3 adaptation machinery. Taken together these results indicate priming adaptation occurs in different CRISPR-Cas systems, that it can be highly active in wild-type strains and that the underlying mechanisms vary.     

A new enzymatic pathway of citrullinogenesis in murine hemopoietic cells

Citrullinogenesis is demonstrated when murine bone marrow cells are incubated with dialyzed secondary mixed leukocyte culture supernatant. The identity of citrulline in bone marrow cell supernatants has been established by gas chromatographic mass spectrometric analysis. It is shown that, in our model, citrulline synthesis proceeds directly from arginine without intermediate ornithine production, ruling out the involvement of ornithine transcarbamylase (EC 2.1.3.3.). Moreover, none of the other enzymatic activities described for catalyzing citrullinogenesis, i.e. arginine deiminase or peptidyl arginine deiminase can be demonstrated. The generation of oxygen radicals is necessary for this enzymatic reaction. It is induced by a thermolabile protein produced during the antiallograft immune response with a molecular weight of about 150,000.     

Effect of neurotensin on pancreatic function in man

The effect of neurotensin on submaximally-stimulated hepatobiliary and pancreatic secretion was studied in 6 healthy subjects. An intravenous infusion of neurotensin 1.4 ± 0.3 pmol/kg/min, designed to reproduce plasma neurotensin immunoreactivity levels within the physiological range, produced a significant increase in pancreatic bicarbonate output. Plasma concentrations of pancreatic polypeptide rose by 83 ± 16 pmol/l and were associated with a small reduction in trypsin, but no significant change in bilirubin outputs.     

Characterization and the broad cross‐species applicability of 20 anonymous nuclear loci isolated from the Taiwan Hwamei (Garrulax taewanus)

We isolated 20 anonymous nuclear loci (8556 bp in total) from the Taiwan Hwamei (Garrulax taewanus), an endemic songbird of Taiwan. A panel of nine to 15 individuals with unknown relationship was used to characterize polymorphism of these loci. We identified 46 single nucleotide polymorphic sites (SNPs) in 15 polymorphic loci. Frequency of SNPs was one per every 186 bp in average. Nucleotide diversity, θ, ranged from 0.00054 to 0.00371 per locus. We also tested cross‐species applicability of these loci on 17 species from eight different passerine families. All 20 loci could be successfully amplified (ranged from one to 16 species, mean = 7.9 species).     

IL-18 enhances IL-4 production by ligand-activated NKT lymphocytes: a pro-Th2 effect of IL-18 exerted through NKT cells

NKT cells are a remarkably versatile population whose functional capacities are determined by cytokines present in their microenvironment. In this study, we provide evidence for a new immunoregulatory effect of the proinflammatory cytokine IL-18 on NKT cells. We found that IL-18, mainly known for its involvement in NK cell activation and in Th 1 immune responses, substantially enhanced IL-4 production as well as the percentage of IL-4(+) cells among NKT lymphocytes activated by their specific ligand alpha-galactosylceramide (alpha-GalCer). The effect of IL-18 on IL-4 production by activated NKT cells took place both in vivo and in vitro and was not affected by IL-12 which increased IFN-gamma secretion in the same conditions. We show that NKT cells are the main targets for IL-18-induced IL-4 production since it occurred neither in NKT-deficient mice nor after stimulation of Th2 lymphocytes. Finally, we provide evidence that the IL-4 promptly generated by NKT cells in response to IL-18 plus alpha-galactosylceramide in vivo can effectively contribute to the adaptive Th2 immune response by up-regulating the early activation marker CD69 on B cells. Our data support the notion that, in contrast to the exclusive IFN-gamma inducer IL-12, IL-18 acts in a more subtle manner as a costimulatory factor in both pro-Th1 and pro-Th2 responses depending on the nature of the stimulation and the target cells.     

Interactions between two cytoskeleton-associated tyrosine kinases: calcium-dependent tyrosine kinase and focal adhesion tyrosine kinase

The calcium-dependent tyrosine kinase (CADTK), also known as Pyk2/RAFTK/CAKbeta/FAK2, is a cytoskeleton-associated tyrosine kinase. We compared CADTK regulation with that of the highly homologous focal adhesion tyrosine kinase (FAK). First, we generated site-specific CADTK mutants. Mutation of Tyr402 eliminated autophosphorylation and significantly decreased kinase activity. Mutation of Tyr881, a putative Src kinase phosphorylation site predicted to bind Grb2, had little effect on CADTK regulation. Src family tyrosine kinases resulted in CADTK tyrosine phosphorylation even when co-expressed with the Tyr402/Tyr881 double mutant, suggesting that Src/Fyn etc. phosphorylate additional tyrosine residues. Interestingly, CADTK tyrosine-phosphorylated FAK when both were transiently expressed, but FAK did not phosphorylate CADTK. Biochemical experiments confirmed direct CADTK phosphorylation of FAK. This phosphorylation utilized tyrosine residues other than Tyr397, Tyr925, or Tyr576/Tyr577, suggesting that new SH2-binding sites might be created by CADTK-dependent FAK phosphorylation. Last, expression of the CADTK carboxyl terminus (CRNK) abolished CADTK but not FAK autophosphorylation. In contrast, FAK carboxyl terminus overexpression inhibited both FAK and CADTK autophosphorylation, suggesting that a FAK-dependent cytoskeletal function may be necessary for CADTK activation. Thus, CADTK and FAK, which both bind to some, but not necessarily the same, cytoskeletal elements, may be involved in coordinate regulation of cytoskeletal structure and signaling.     

A distinct IL-18-induced pathway to fully activate NK T lymphocytes independently from TCR engagement

NK T lymphocytes are characterized by their ability to promptly generate IL-4 and IFN-gamma upon TCR engagement. Here, we demonstrate that these cells can also be fully activated in the absence of TCR cross-linking in response to the proinflammatory cytokine IL-18 associated with IL-12. NK T cells stimulated with IL-18 plus IL-12 proliferated, killed Fas+ target cells, and produced high levels of IFN-gamma without IL-4. In these conditions, IFN-gamma production was at least 10-fold higher than that upon TCR cross-linking. Interestingly, a 2-h pretreatment with IL-12 plus IL-18 sufficed to maintain the high IFN-gamma-producing potential during subsequent stimulation with anti-TCR mAbs or with the specific Ag alpha-galactosylceramide. Similar effects were observed in vivo, because splenic CD4+ NK T cells from MHC class II-deficient mice secreted IFN-gamma without further stimulation when removed 2 h after a single injection of IL-12 plus IL-18. In conclusion, our evidence for activation of NK T lymphocytes in response to IL-18 plus IL-12 in the absence of TCR engagement together with the maintenance of preferential IFN-gamma vs IL-4 production upon subsequent exposure to specific Ags is consistent with the active participation of this cell population in innate as well as acquired cellular immune responses.