Selective and specific internalization of clostridial C3 ADP‐ribosyltransferases into macrophages and monocytes

The C3 transferases from Clostridium botulinum (C3bot) and Clostridium limosum (C3lim) mono‐ADP‐ribosylate and thereby inactivate RhoA, ‐B and ‐C of eukaryotic cells. Due to their extremely poor cellular uptake, C3 transferases were supposed to be exoenzymes rather than exotoxins, challenging their role in pathogenesis. Here, we report for the first time that low concentrations of both C3lim and C3bot are selectively internalized into macrophages/monocytes in less than 3 h, inducing the reorganization of the actin cytoskeleton by ADP‐ribosylation of Rho. We demonstrate that C3 transferases are internalized into the cytosol of macrophages/monocytes via acidified early endosomes. Bafilomycin A1, an inhibitor of endosomal acidification, protected J774A.1 macrophages and human promyelotic leukaemia cells (HL‐60) from intoxication by C3. Moreover, confocal laser scanning microscopy revealed colocalization of C3 with early endosomes. An extracellular acidic pulse enabled direct translocation of cell surface‐bound C3 across the cytoplasmic membrane to the cytosol. In line with this finding, both C3 proteins exhibited membrane activity in lipid bilayer membranes only under acidic conditions (pH < 5.5). In conclusion, we identified macrophages/monocytes as target cells for clostridial C3 transferases and shed light on their selective uptake mechanism, which might contribute to understand the role of C3 transferases in pathogenesis.     

Acetylcholine receptor pathway mutations explain various fetal akinesia deformation sequence disorders

Impaired fetal movement causes malformations, summarized as fetal akinesia deformation sequence (FADS), and is triggered by environmental and genetic factors. Acetylcholine receptor (AChR) components are suspects because mutations in the fetally expressed gamma subunit (CHRNG) of AChR were found in two FADS disorders, lethal multiple pterygium syndrome (LMPS) and Escobar syndrome. Other AChR subunits alpha1, beta1, and delta (CHRNA1, CHRNB1, CHRND) as well as receptor-associated protein of the synapse (RAPSN) previously revealed missense or compound nonsense-missense mutations in viable congenital myasthenic syndrome; lethality of homozygous null mutations was predicted but never shown. We provide the first report to our knowledge of homozygous nonsense mutations in CHRNA1 and CHRND and show that they were lethal, whereas novel recessive missense mutations in RAPSN caused a severe but not necessarily lethal phenotype. To elucidate disease-associated malformations such as frequent abortions, fetal edema, cystic hygroma, or cardiac defects, we studied Chrna1, Chrnb1, Chrnd, Chrng, and Rapsn in mouse embryos and found expression in skeletal muscles but also in early somite development. This indicates that early developmental defects might be due to somite expression in addition to solely muscle-specific effects. We conclude that complete or severe functional disruption of fetal AChR causes lethal multiple pterygium syndrome whereas milder alterations result in fetal hypokinesia with inborn contractures or a myasthenic syndrome later in life.     

Abscisic acid induces somatic embryogenesis and enables the capture of high-value genotypes in Douglas fir (Pseudotsuga menziesii [MIRB.] Franco)

Douglas fir (Pseudotsuga menziesii) is one of Europe’s most important non-native tree species due to its drought tolerance as well as timber quality and yield. To obtain superior seed from selected parental trees, breeding programs had been established in seed orchards. Douglas fir seed is used as source material for somatic embryogenesis with the aim to select elite genotypes invaluable for clonal mass propagation. To improve given protocols for somatic embryo initiation, we used immature Douglas fir zygotic embryos as explants and abscisic acid (ABA) as plant growth regulator in contrast to the application of auxins and cytokinins. With ABA supplementation, induction frequencies were slightly but in mean higher than with auxin/cytokinin, showing also a strong genotype effect. This offered the possibility to capture SE cultures from otherwise recalcitrant crosses. Furthermore, we observed remarkable differences between the two sets of plant growth regulators concerning the morphological development of the explants, including the absence of non-embryogenic callus by using ABA as inducer. This simplifies the detection of events and the handling of the obtained cultures. Nevertheless, a histological approach suggested, that the same competent cells are addressed by the different hormonal stimulation. Besides, we studied the influence of different points in time of cone harvest, two different basal media and different genetic backgrounds of the explants as well as the maturation ability of the induced embryogenic cultures. In sum, we were able to improve the first steps of somatic embryogenesis and to maintain a significantly higher number of high-value genotypes.