Site-selective glycosylation of proteins: creating synthetic glycoproteins

In higher organisms, the functions of many proteins are modulated by post-translational modifications (PTMs). Glycosylation is by far the most diverse of the PTM processes. Natural protein production methods typically produce PTM or glycoform mixtures within which function is difficult to dissect or control. Chemical tagging methods allow the precise attachment of multiple glycosylation modifications to bacterially expressed (bare) protein scaffolds, allowing reconstitution of functionally effective mimics of glycoproteins in higher organisms. In this way combining chemical control of PTM with readily available protein scaffolds provides a systematic platform for creating probes of protein-PTM interactions. This protocol describes the modification of Cys residues in proteins using glycomethanethiosulfonates and glycoselenenylsulfides and the modification of azidohomoalanine residues, introduced by Met replacement using auxotrophic Met(-) Escherichia coli strains, with glycoalkynes and the combination of these techniques for the creation of dual-tagged proteins. Each glycosylation procedure outlined in this protocol can be achieved in half a day.     

On the origin of the term "stem cell"

Stem cells have fascinated both biologists and clinicians for over a century. Here, we discuss the origin of the term "stem cell," which can be traced back to the late 19th century. The term stem cell originated in the context of two major embryological questions of that time: the continuity of the germ-plasm and the origin of the hematopoietic system. Theodor Boveri and Valentin H?cker used the term stem cell to describe cells committed to give rise to the germline. In parallel, Artur Pappenheim, Alexander Maximow, Ernst Neumann, and others used it to describe a proposed progenitor of the blood system. The original meanings of the term stem cell, rather than being historical relics, continue to capture important aspects of the biology of stem cells as we see them today.     

Structural and functional evolution of the P2Y12-like receptor group

Metabotropic pyrimidine and purine nucleotide receptors (P2Y receptors) belong to the superfamily of G protein-coupled receptors (GPCR). They are distinguishable from adenosine receptors (P1) as they bind adenine and/or uracil nucleotide triphosphates or diphosphates depending on the subtype. Over the past decade, P2Y receptors have been cloned from a variety of tissues and species, and as many as eight functional subtypes have been characterized. Most recently, several members of the P2Y₁₂-like receptor group, which includes the clopidogrel-sensitive ADP receptor P2Y₁₂, have been deorphanized. The P2Y₁₂-like receptor group comprises several structurally related GPCR which, however, display heterogeneous agonist specificity including nucleotides, their derivatives, and lipids. Besides the established function of P2Y₁₂ in platelet activation, expression in macrophages, neuronal and glial cells as well as recent results from functional studies implicate that several members of this group may have specific functions in neurotransmission, inflammation, chemotaxis, and response to tissue injury. This review focuses specifically on the structure-function relation and shortly summarizes some aspects of the physiological relevance of P2Y₁₂-like receptor members.     

Interaction of statins with phospholipid bilayers studied by solid-state NMR spectroscopy

Statins are drugs that specifically inhibit the enzyme HMG-CoA reductase and thereby reduce the concentration of low-density lipoprotein cholesterol, which represents a well-established risk factor for the development of atherosclerosis. The results of several clinical trials have shown that there are important intermolecular differences responsible for the broader pharmacologic actions of statins, even beyond HMG-CoA reductase inhibition. According to one hypothesis, the biological effects exerted by these compounds depend on their localization in the cellular membrane. The aim of the current work was to study the interactions of different statins with phospholipid membranes and to investigate their influence on the membrane structure and dynamics using various solid-state NMR techniques. Using ¹H NOESY MAS NMR, it was shown that atorvastatin, cerivastatin, fluvastatin, rosuvastatin, and some percentage of pravastatin intercalate the lipid-water interface of POPC membranes to different degrees. Based on cross-relaxation rates, the different average distribution of the individual statins in the bilayer was determined quantitatively. Investigation of the influence of the investigated statins on membrane structure revealed that lovastatin had the least effect on lipid packing and chain order, pravastatin significantly lowered lipid chain order, while the other statins slightly decreased lipid chain order parameters mostly in the middle segments of the phospholipid chains.     

Development of a bioassay to assess resistance to Fusarium oxysporum (Schlecht.) in asparagus (Asparagus officinalis L.)

Fusarium oxysporum is one of the major pathogens causing root and crown rot in asparagus. Breeding of cultivars resistant to F. oxysporum would be the most efficient strategy for pathogen control. In this study, a bioassay was developed for screening seedling resistance. The non‐destructive bioassay comprises inoculation with a highly aggressive F. oxysporum isolate, incubation in a climate chamber and quantification of disease symptoms by a digital image analysing system and a PTA‐ELISA. This bioassay is simple to implement and demonstrated high reproducibility. Subsequently, it was used to determine the resistance behaviour of 16 asparagus genotypes to F. oxysporum. The asparagus cultivars revealed different levels of susceptibility, whereas the wild relative A. densiflorus was confirmed to be resistant.     

Preliminary survey on putative insect vectors for Rubus stunt phytoplasmas

Phytoplasmas are cell wall‐less phytopathogenic bacteria which are associated with a disease in Rubus species known as Rubus stunt. Symptoms range from stunting, witches’ broom, small leaves, short internodes, enlarged sepals, phyllody and flower proliferation to fruit malformations. Phytoplasmas can be spread by vegetative propagation and by phloem‐feeding insect vectors. However, little is known about the spectrum and distribution of putative Rubus stunt insect vectors. In this study, a screening of putative insect vectors of Rubus stunt in raspberry plantations in southern and northern Germany was carried out during two successive years (2014 and 2015) with multiple sampling dates throughout the growing seasons. A total of 2,891 hemipteran insects were sorted, identified to family, genus or species level when possible, and a subset of 319 DNA samples containing a sum of 932 selected individuals representing all identified species, sampling locations and sampling dates were tested for phytoplasma DNA using qPCR. Altogether, eight DNA samples were positive for phytoplasma DNA, among them species from the genera Euscelidius, Macrosteles, Euscelis, Anaceratagalliaand Psammotettix. These data will form the basis for choosing and timing appropriate control measures against Rubus stunt and also for potential insect vector transmission experiments.