Detection of Bacterial α-l-Fucosidases with an Ortho-Quinone Methide-Based Probe and Mapping of the Probe-Protein Adducts.

Fucosidases are associated with several pathological conditions and play an important role in the health of the human gut. For example, fucosidases have been shown to be indicators and/or involved in hepatocellular carcinoma, breast cancer, and helicobacter pylori infections. A prerequisite for the detection and profiling of fucosidases is the formation of a specific covalent linkage between the enzyme of interest and the activity-based probe (ABP). The most commonly used fucosidase ABPs are limited to only one of the classes of fucosidases, the retaining fucosidases. New approaches are needed that allow for the detection of the second class of fucosidases, the inverting type. Here, we report an ortho-quinone methide-based probe with an azide mini-tag that selectively labels both retaining and inverting bacterial α-l-fucosidases. Mass spectrometry-based intact protein and sequence analysis of a probe-labeled bacterial fucosidase revealed almost exclusive single labeling at two specific tryptophan residues outside of the active site. Furthermore, the probe could detect and image extracellular fucosidase activity on the surface of live bacteria.

Trafficking of MHC class II in dendritic cells is dependent on but not regulated by degradation of its associated invariant chain.

In dendritic cells (DC), newly synthesized MHCII is directed to endosomes by its associated invariant chain (Ii). Here, Ii is degraded after which MHCII is loaded with peptides. In immature DC, ubiquitination of peptide-loaded MHCII drives its sorting to lysosomes for degradation. Ubiquitination of MHCII is strongly reduced in response to inflammatory stimuli, resulting in increased expression of MHCII at the plasma membrane. Whether surface exposure of MHCII is also regulated during DC maturation by changing the rate of Ii degradation remained unresolved by conflicting results in the literature. We here pinpoint experimental problems that have contributed to these controversies and demonstrate that immature and mature DC degrade Ii equally efficient at proper culture conditions. Only when DC were cultured in glutamine containing media, endosome acidification and Ii degradation were restricted in immature DC and enhanced in response to lipopolysaccharide (LPS). These effects are caused by ammonia, a glutamine decomposition product. This artificial behavior could be prevented by culturing DC in media containing a stable dipeptide as glutamine source. We conclude that Ii degradation is a prerequisite for but not a rate limiting step in MHCII processing.

Evaluation of radiographic and genetic aspects of hereditary subluxation of the radial head in Bouviers des Flandres.

OBJECTIVE: To study radiographic and genetic aspects of hereditary radial head subluxation in Bouviers des Flandres. ANIMALS: 26 related Bouviers des Flandres affected with bilateral subluxation of the radial head, 10 unaffected related dogs, and 29 unrelated Bouviers des Flandres with diagnoses of nonskeletal diseases. PROCEDURES: All dogs were radiographically studied, and their DNA was analyzed with a genome-wide screen of 1,536 single nucleotide polymorphisms. In addition, karyotyping was performed in an unaffected dam and its affected offspring. RESULTS: Both forelimbs of affected dogs were disproportionately short with caudolateral subluxation or luxation of the radial head. Angulation of the radial axis at the mid-diaphysis ranged from 9.3 degrees to 30.3 degrees (mean +/- SD, 14.9 +/- 6.1 degrees ), with an estimated age of onset from 0 to 4 months. Poorly defined medial coronoid processes and osteoarthritis of the elbow joint, cranial bowing of the olecranon, and disturbed growth in length of the ulna with sharply demarcated spurs were noticed on radiographs of affected dogs. Genealogical analysis indicated that most affected dogs were closely related, but the mode of inheritance was not clear. The DNA analysis found that 205 single nucleotide polymorphisms were monomorphic in the affected dogs. Conventional chromosome staining revealed no numerical chromosomal aberration. CONCLUSIONS AND CLINICAL RELEVANCE: Congenital radial head luxation and subluxation in the studied Bouviers des Flandres were characterized by angulation of the radial axis leading to caudolateral subluxation of the radial head and insufficient growth of the distal portion of the ulna together with cranial bowing of the olecranon.

Dendritic cells release exosomes together with phagocytosed pathogen; potential implications for the role of exosomes in antigen presentation.

Dendritic cells (DC) have the unique capacity to activate naïve T cells by presenting T cell receptor specific peptides from exogenously acquired antigens bound to Major Histocompatibility Complex (MHC) molecules. MHC molecules are displayed on the DC plasma membrane as well as on extracellular vesicles (EV) that are released by DC, and both have antigen-presenting capacities. However, the physiological role of antigen presentation by EV is still unclear. We here demonstrate that the release of small EV by activated DC is strongly stimulated by phagocytic events. We show that, concomitant with the enhanced release of EV, a significant proportion of phagocytosed bacteria was expulsed back into the medium. High-resolution fluorescence microscopic images revealed that bacteria in phagosomes were surrounded by EV marker-proteins. Moreover, expulsed bacteria were often found associated with clustered HLA II and CD63. Together, these observations suggest that exosomes may be formed by the inward budding into phagosomes, whereupon they are secreted together with the phagosomal content. These findings may have important implications for selective loading of peptides derived from phagocytosed pathogens onto exosome associated HLA molecules, and have important implications for vaccine design.

Bystander T-Cells Support Clonal T-Cell Activation by Controlling the Release of Dendritic Cell-Derived Immune-Stimulatory Extracellular Vesicles.

Extracellular vesicles (EV) that are released by immune cells are studied intensively for their functions in immune regulation and are scrutinized for their potential in human immunotherapy, for example against cancer. In our search for signals that stimulate the release of functional EV by dendritic cells we observed that LPS-activated human monocyte-derived dendritic cells (moDC) changed their morphological characteristics upon contact with non-cognate activated bystander T-cells, while non-activated bystander T-cells had no effect. Exposure to activated bystander T-cells also stimulated the release of EV-associated proteins by moDC, particularly CD63, and ICAM-1, although the extent of stimulation varied between individual donors. Stimulation of moDC with activated bystander T-cells also increased the release of EV-associated miR155, which is a known central modulator of T-cell responses. Functionally, we observed that EV from moDC that were licensed by activated bystander T-cells exhibited a capacity for antigen-specific T-cell activation. Taken together, these results suggest that non-cognatei interactions between DC and bystander T-cells modulates third party antigen-specific T-cell responses via EV.

Organization and concerted evolution of the ampliconic Y-chromosomal TSPY genes from cattle.

The Y-chromosomal gene TSPY (testis-specific protein Y-encoded) is probably involved in early spermatogenesis and has a variable copy number in different mammalian species. Analysis of bovine BAC clones leads to an estimate of 90 TSPY loci on the bovine Y chromosome. Half of these loci (TSPY-M1 and TSPY-M2) contain a single copy, while the other loci (TSPY-C) contain a cluster of three, possibly four, truncated pseudogenes. Fluorescence in situ hybridization indicated that the TSPY loci are located mainly on the short arm (Yp). The TSPY genes appear to account for about 2.5% of the Y chromosome and contain several published bovine Y-chromosomal microsatellites. The homology of TSPY and the major Y-chromosomal repetitive elements BRY.2 from cattle and OY.1 from sheep (80-85% similarity) further illustrates how the Y chromosome is shaped by rearrangements and horizontal spreading of the most abundant sequences. A comparison of TSPY-M1 sequences from different BAC clones and from related bovine species suggests concerted evolution as one of the mechanisms of the rapid evolution of the mammalian Y chromosome.