Immunogenicity Studies with Haptenated Melittin Peptides: Implication for Membrane Involvement during the Recognition Step

Melittin peptides carrying 2,4-dinitro-6-carboxyphenyl (Dncp) haptenic groups regularly evoked anti-hapten IgG responses in mice or guinea pigs when the hapten was C-terminally attached. Single haptens on the N-terminal helix in several positions gave poor or no responses in the early stages but adequate titres after prolonged immunization. Peptides with Dncp at the C-terminus as an invariant feature and a second Dncp in various positions along the peptide chain did not fail to produce adequate responses. The hampering effect is not due to a defect at the T-cell level but involves the recognition step on the B-cell. It is implied that the haptenic interaction with the paratope of the recognizing immunoglob ulin on the B-cell involves the cell membrane in an important way. It is also suggested that late antibody responses should not be overlooked during the development of proteinaceous immunogens for vaccination.     

Phagocytosis in Dictyostelium: Nibbling, Eating and Cannibalism

ABSTRACT. Phagocytosis is a highly conserved biological process that serves numerous functions in a wide variety of organisms. Over the past few decades Dictyostelium has proven to be an excellent organism for investigations in cell biology and this is certainly no less the case for a study of phagocytosis. This review examines three distinct phagocytic activities which have been characterized in Dictyostelium. The first, "vegetative phagocytosis," represents the classical eukaryotic microbial uptake of food particles (bacteria). The second, a predatory form of phagocytosis, arises when one species such as Dictyostelium caveatum attacks another species of slime mold, engulfing small pieces of the target prey. This has been termed "cell nibbling." The third phagocytic process is "sexual cannibalistic phagocytosis." In this situation a zygote giant cell, having arisen from the fusion of gametic amoebae, attracts unfused nonzygotic amoebae of the same species and engulfs them as a food source. While cell nibbling has not been actively studied, vegetative and sexual cannibalistic phagocytosis have received varying amounts of attention leading to the idea that some of the elements (e.g., glycoprotein receptors and a G_αs subunit) involved in certain of these phagocytic events may be the same. On the other hand, some unique events (e.g., filopodial induction in prey by D. caveatum ) are also worthy of further investigation. Among other things, the presence of self-nonself recognition, the existence of opsonin-like substances and the presence of signal transduction elements (e.g., an A2-like receptor that negatively modulates sexual phagocytosis) once considered to be extant only in higher organisms suggest that much can be learned about phagocytosis in general by further studies in the classic, eukaryotic microbe Dictyostelium discoideum and related species.     

Arabinose 5-phosphate isomerase as a target for antibacterial design: Studies with substrate analogues and inhibitors

Structural requirements of d-arabinose 5-phosphate isomerase (KdsD, E.C. 5.3.1.13) from Pseudomonas aeruginosa were analysed in detail using advanced NMR techniques. We performed epitope mapping studies of the binding between the enzyme and the most potent KdsD inhibitors found to date, together with studies of a set of newly synthesised arabinose 5-phosphate (A5P) mimetics. We report here the first experimental evidence that KdsD may bind the furanose form of A5P, suggesting that catalysis of ring opening may be an important part of KdsD catalysis.     

A New Monoclonal Antibody Enables BAR Analysis of Subcellular Importin β1 Interactomes

Importin β1 (KPNB1) is a nucleocytoplasmic transport factor with critical roles in both cytoplasmic and nucleocytoplasmic transport, hence there is keen interest in the characterization of its subcellular interactomes. We found limited efficiency of BioID in the detection of importin complex cargos and therefore generated a highly specific and sensitive anti-KPNB1 monoclonal antibody to enable biotinylation by antibody recognition analysis of importin β1 interactomes. The monoclonal antibody recognizes an epitope comprising residues 301-320 of human KPBN1 and strikingly is highly specific for cytoplasmic KPNB1 in diverse applications, with little reaction with KPNB1 in the nucleus. Biotinylation by antibody recognition with this novel antibody revealed numerous new interactors of importin β1, expanding the KPNB1 interactome to cytoplasmic and signaling complexes that highlight potential new functions for the importins complex beyond nucleocytoplasmic transport. Data are available via ProteomeXchange with identifier PXD032728.     

Deciphering Spatial Protein–Protein Interactions in Brain Using Proximity Labeling

Cellular biomolecular complexes including protein–protein, protein–RNA, and protein–DNA interactions regulate and execute most biological functions. In particular in brain, protein–protein interactions (PPIs) mediate or regulate virtually all nerve cell functions, such as neurotransmission, cell–cell communication, neurogenesis, synaptogenesis, and synaptic plasticity. Perturbations of PPIs in specific subsets of neurons and glia are thought to underly a majority of neurobiological disorders. Therefore, understanding biological functions at a cellular level requires a reasonably complete catalog of all physical interactions between proteins. An enzyme-catalyzed method to biotinylate proximal interacting proteins within 10 to 300 nm of each other is being increasingly used to characterize the spatiotemporal features of complex PPIs in brain. Thus, proximity labeling has emerged recently as a powerful tool to identify proteomes in distinct cell types in brain as well as proteomes and PPIs in structures difficult to isolate, such as the synaptic cleft, axonal projections, or astrocyte–neuron junctions. In this review, we summarize recent advances in proximity labeling methods and their application to neurobiology.     

Behavioral signatures of structured feature detection during courtship in Drosophila

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Ribonucleotides as nucleotide excision repair substrates

The incorporation of ribonucleotides in DNA has attracted considerable notice in recent years, since the pool of ribonucleotides can exceed that of the deoxyribonucleotides by at least 10–20-fold, and single ribonucleotide incorporation by DNA polymerases appears to be a common event. Moreover ribonucleotides are potentially mutagenic and lead to genome instability. As a consequence, errantly incorporated ribonucleotides are rapidly repaired in a process dependent upon RNase H enzymes. On the other hand, global genomic nucleotide excision repair (NER) in prokaryotes and eukaryotes removes damage caused by covalent modifications that typically distort and destabilize DNA through the production of lesions derived from bulky chemical carcinogens, such as polycyclic aromatic hydrocarbon metabolites, or via crosslinking. However, a recent study challenges this lesion-recognition paradigm. The work of Vaisman et al. (2013) [34] reveals that even a single ribonucleotide embedded in a deoxyribonucleotide duplex is recognized by the bacterial NER machinery in vitro. In their report, the authors show that spontaneous mutagenesis promoted by a steric-gate pol V mutant increases in uvrA, uvrB, or uvrC strains lacking rnhB (encoding RNase HII) and to a greater extent in an NER-deficient strain lacking both RNase HI and RNase HII. Using purified UvrA, UvrB, and UvrC proteins in in vitro assays they show that despite causing little distortion, a single ribonucleotide embedded in a DNA duplex is recognized and doubly-incised by the NER complex. We present the hypothesis to explain the recognition and/or verification of this small lesion, that the critical 2′-OH of the ribonucleotide – with its unique electrostatic and hydrogen bonding properties – may act as a signal through interactions with amino acid residues of the prokaryotic NER complex that are not possible with DNA. Such a mechanism might also be relevant if it were demonstrated that the eukaryotic NER machinery likewise incises an embedded ribonucleotide in DNA.     

The effect of temperature on progeny allocation and short interval timing in a parasitoid wasp

Abstract The parasitoid wasp Trichogramma dendrolimi Matsumura sets the number of progeny allocated to its insect egg hosts according to the duration of its initial transit walk across the host surface. Although cooling the wasp reduces its walking speed, reduced temperature does not affect progeny allocation. By locally heating and cooling the wasps, the initial transit can be thermally uncoupled from the subsequent oviposition. Using this technique we show that the timing of initial transit duration is temperature-dependent. These findings suggest that short interval timing by Trichogramma differs physiologically from the temperature compensated clocks that have been described for other insects.