Systematic Analysis of proteoglycan modification sites in Caenorhabditis elegans by scanning mutagenesis

Proteoglycan modification is essential for development and early cell division in Caenorhabditis elegans. The specification of proteoglycan attachment sites is defined by the Golgi enzyme polypeptide xylosyltransferase. Here we evaluate the substrate specificity of this xylosyltransferase for its downstream targets by using reporter proteins containing proteoglycan modification sites from C. elegans syndecan/SDN-1. The N terminus of the SDN-1 contains a Ser-Gly proteoglycan site at Ser(71), flanked by potential mucin and N-glycosylation sites. However, Ser(71) was exclusively used as a proteoglycan site in vivo, based on mapping studies with a Ser(71) reporter protein, glycosyltransferase RNA interference, and co-expression of worm polypeptide xylosyltransferase. To elucidate the substrate requirements of this enzyme, a library of 42 point mutants of the Ser(71) reporter was expressed in tissue culture. The nematode proteoglycan modification site in SDN-1 required serine (not threonine), two flanking glycine residues (positions -1 and +1), and either one proximal acidic N-terminal amino acid (positions -4, -3, and -2) or a pair of distal N-terminal acidic amino acids (positions -6 and -5). C-terminal acidic amino acids, although present in many proteoglycan modification sites, had minimal impact on xylosylation at Ser(71). Proline inhibited glycosylation when present at -1, +1, or +2. The position of glycine, proline, and acidic amino acids allows the glycosylation machinery to discriminate between mucin and proteoglycan modification sites. The key residues that define proteoglycan modification sites also function with the Drosophila polypeptide xylosyltransferase, indicating that the specificity in the glycosylation process is evolutionarily conserved. Using a neural network method, a preliminary proteoglycan predictor has been developed.     

Yttrium 90 ibritumomab tiuxetan (Zevalin®): A new bullet in the fight against malignant lymphoma?

Targeted immunotherapy utilizing monoclonal antibodies has improved survival in both indolent and aggressive lymphoma significantly. Since malignant lymphomas are also responsive to radiation, the combination of targeted immunotherapy and radiation with radionuclide coupled to monoclonal antibodies is an attractive option to further improve treatment results. Zevalin is a commercially available variation of the murine anti-CD20 antibody ibritumomab coupled to Yttrium 90 via the tiuxetan chelator. The development of this novel drug as well as the results of the pivotal studies and clinical strategies to implement this treatment into the routine practice of lymphoma oncology are presented in this review.     

Age- and sex-specific mortality patterns in an emerging wildlife epidemic: the phocine distemper in European harbour seals

Analyses of the dynamics of diseases in wild populations typically assume all individuals to be identical. However, profound effects on the long-term impact on the host population can be expected if the disease has age and sex dependent dynamics. The Phocine Distemper Virus (PDV) caused two mass mortalities in European harbour seals in 1988 and in 2002. We show the mortality patterns were highly age specific on both occasions, where young of the year and adult (>4 yrs) animals suffered extremely high mortality, and sub-adult seals (1-3 yrs) of both sexes experienced low mortality. Consequently, genetic differences cannot have played a main role explaining why some seals survived and some did not in the study region, since parents had higher mortality levels than their progeny. Furthermore, there was a conspicuous absence of animals older than 14 years among the victims in 2002, which strongly indicates that the survivors from the previous disease outbreak in 1988 had acquired and maintained immunity to PDV. These specific mortality patterns imply that contact rates and susceptibility to the disease are strongly age and sex dependent variables, underlining the need for structured epidemic models for wildlife diseases. Detailed data can thus provide crucial information about a number of vital parameters such as functional herd immunity. One of many future challenges in understanding the epidemiology of the PDV and other wildlife diseases is to reveal how immune system responses differ among animals in different stages during their life cycle. The influence of such underlying mechanisms may also explain the limited evidence for abrupt disease thresholds in wild populations.     

Syk-mediated translocation of PI3Kdelta to the leading edge controls lamellipodium formation and migration of leukocytes

The non-receptor tyrosine kinase Syk is mainly expressed in the hematopoietic system and plays an essential role in beta(2) integrin-mediated leukocyte activation. To elucidate the signaling pathway downstream of Syk during beta2 integrin (CD11/CD18)-mediated migration and extravasation of polymorphonuclear neutrophils (PMN), we generated neutrophil-like differentiated HL-60 (dHL-60) cells expressing a fluorescently tagged Syk mutant lacking the tyrosine residue at the position 323 (Syk-Tyr323) that is known to be required for the binding of the regulatory subunit p85 of the phosphatidylinositol 3-kinase (PI3K) class I(A). Syk-Tyr323 was found to be critical for the enrichment of the catalytic subunit p110delta of PI3K class I(A) as well as for the generation of PI3K products at the leading edge of the majority of polarized cells. In accordance, the translocation of PI3K p110delta to the leading edge was diminished in Syk deficient murine PMN. Moreover, the expression of EGFP-Syk Y323F interfered with proper cell polarization and it impaired efficient migration of dHL-60 cells. In agreement with a major role of beta2 integrins in the recruitment of phagocytic cells to sites of lesion, mice with a Syk-deficient hematopoietic system demonstrated impaired PMN infiltration into the wounded tissue that was associated with prolonged cutaneous wound healing. These data imply a novel role of Syk via PI3K p110delta signaling for beta2 integrin-mediated migration which is a prerequisite for efficient PMN recruitment in vivo.     

Protein A chromatography for antibody purification

Staphylococcal protein A (SPA) is one of the first discovered immunoglobulin binding molecules and has been extensively studied during the past decades. Due to its affinity to immunoglobulins, SPA has found widespread use as a tool in the detection and purification of antibodies and the molecule has been further developed to one of the most employed affinity purification systems. Interestingly, a minimized SPA derivative has been constructed and a domain originating from SPA has been improved to withstand the harsh environment employed in industrial purifications. This review will focus on the development of different affinity molecules and matrices for usage in antibody purification.     

Characterization of reemerging chikungunya virus

An unprecedented epidemic of chikungunya virus (CHIKV) infection recently started in countries of the Indian Ocean area, causing an acute and painful syndrome with strong fever, asthenia, skin rash, polyarthritis, and lethal cases of encephalitis. The basis for chikungunya disease and the tropism of CHIKV remain unknown. Here, we describe the replication characteristics of recent clinical CHIKV strains. Human epithelial and endothelial cells, primary fibroblasts and, to a lesser extent, monocyte-derived macrophages, were susceptible to infection and allowed viral production. In contrast, CHIKV did not replicate in lymphoid and monocytoid cell lines, primary lymphocytes and monocytes, or monocyte-derived dendritic cells. CHIKV replication was cytopathic and associated with an induction of apoptosis in infected cells. Chloroquine, bafilomycin-A1, and short hairpin RNAs against dynamin-2 inhibited viral production, indicating that viral entry occurs through pH-dependent endocytosis. CHIKV was highly sensitive to the antiviral activity of type I and II interferons. These results provide a general insight into the interaction between CHIKV and its mammalian host.     

Specialized mismatch repair function of Glu339 in the Phe-X-Glu motif of yeast Msh6

The major eukaryotic mismatch repair (MMR) pathway requires Msh2-Msh6, which, like Escherichia coli MutS, binds to and participates in repair of the two most common replication errors, single base-base and single base insertion-deletion mismatches. For both types of mismatches, the side chain of E. coli Glu38 in a conserved Phe-X-Glu motif interacts with a mismatched base. The Ovarepsilon of Glu38 forms a hydrogen bond with either the N7 of purines or the N3 of pyrimidines. We show here that changing E. coli Glu38 to alanine results in nearly complete loss of repair of both single base-base and single base deletion mismatches. In contrast, a yeast strain with alanine replacing homologous Glu339 in Msh6 has nearly normal repair for insertion-deletion and most base-base mismatches, but is defective in repairing base-base mismatches characteristic of oxidative stress, e.g. 8-oxo-G.A mismatches. The results suggest that bacterial MutS and yeast Msh2-Msh6 differ in how they recognize and/or process replication errors involving undamaged bases, and that Glu339 in Msh6 may have a specialized role in repairing mismatches containing oxidized bases.