Take the tube: remodelling of the endosomal system by intracellular Salmonella enterica

Salmonella enterica is a facultative intracellular pathogen residing in a unique host cell‐derived membrane compartment, termed Salmonella‐containing vacuole or SCV. By the activity of effector proteins translocated by the SPI2‐endoced type III secretion system (T3SS), the biogenesis of the SCV is manipulated to generate a habitat permissive for intracellular proliferation. By taking control of the host cell vesicle fusion machinery, intracellular Salmonella creates an extensive interconnected system of tubular membranes arising from vesicles of various origins, collectively termed Salmonella‐induced tubules (SIT). Recent work investigated the dynamic properties of these manipulations. New host cell targets of SPI2‐T3SS effector proteins were identified. By applying combinations of live cell imaging and ultrastructural analyses, the detailed organization of membrane compartments inhabited and modified by intracellular Salmonella is now available. These studies provided unexpected new details on the intracellular environments of Salmonella. For example, one kind of SIT, the LAMP1‐positive Salmonella‐induced filaments (SIF), are composed of double‐membrane tubules, with an inner lumen containing host cell cytosol and cytoskeletal filaments, and an outer lumen containing endocytosed cargo. The novel findings call for new models for the biogenesis of SCV and SIT and give raise to many open questions we discuss in this review.     

Chiral superstructures of insulin amyloid fibrils

Hydrodynamic forces are capable of inducing structural order in dispersed solid phases, and of causing symmetry-breaking when chiral crystals precipitate from an achiral liquid phase. Until it was observed upon vortex-assisted fibrillation of insulin, such behavior had been thought to be confined to few unbiological systems. In this paper we are discussing chiroptical properties of two chiral variants of insulin amyloid, termed +ICD and -ICD, which form during the process of chiral bifurcation in vortexed solutions of aggregating insulin. As conventional measurements of circular dichroism of solid, anisotropic substances are particularly vulnerable to overlapping influences of linear birefringence and linear dichroism, we have employed complementary tools including dedicated universal chiroptical spectrophotometer to rule out such artifacts. We propose that the strong chiroptical properties of +ICD and -ICD insulin fibrils are an aspect of genuine superstructural chirality of amyloid fibrils and of powerful excitonic couplings taking place within them. A comparison of thioflavin T complexes with fibrils formed by insulin and polyglutamic acid suggests that the extrinsic Cotton effect stemming from the level of single twisted dye molecules is weaker, although diagnostically useful, and cannot account for the overall magnitude of ICD of the dye bound to ±ICD insulin amyloid.     

Identification of adult mineralized tissue zebrafish mutants

Zebrafish craniofacial, skeletal, and tooth development closely resembles that of higher vertebrates. Our goal is to identify viable adult zebrafish mutants that can be used as models for human mineralized craniofacial, dental, and skeletal system disorders. We used a large-scale forward-genetic chemical N-ethyl-nitroso-urea mutagenesis screen to identify 17 early lethal homozygous recessive mutants with defects in craniofacial cartilage elements, and 7 adult homozygous recessive mutants with mineralized tissue phenotypes including craniofacial shape defects, fused sutures, dysmorphic or missing skeletal elements, scoliosis, and neural arch defects. One mutant displayed both an early lethal homozygous phenotype and an adult heterozygous phenotype. These results extend the utility of the zebrafish model beyond the embryo to study human bone and cartilage disorders.     

Effects of maternal stress on egg characteristics in a cooperatively breeding fish

Elevated stress experienced by a mother can compromise both her own reproductive success and that of her offspring. In this study, we investigated whether chronically stressed mothers experienced such effects in cooperatively breeding species, in which helpers at the nest potentially compound the negative effects of maternal stress. Using Neolamprologus pulcher, a group-living cichlid fish from Lake Tanganyika, we observed the effects of experimentally increased stress on female reproductive success (measured as inter-spawn interval, and number of eggs) as well as egg characteristics including egg size and cortisol concentrations. Stress levels were manipulated by repeated exposure to the acute stresses of chasing and netting. Stressed females had longer inter-spawn intervals and laid fewer, smaller eggs. Although no significant differences in egg cortisol concentrations were detected between control and stressed females, egg cortisol concentration fell between spawns in control but not in stressed fish. No effect of helper number was detected for any parameter examined, except there appeared to be less change in egg cortisol content in groups with helpers present. Our results suggest that stress imposes fitness costs on breeding females, and social regulation of a dominance hierarchy does not appear to exacerbate or alleviate the negative effects of maternal stress.     

Odorant-dependent, spatially restricted induction of c-fos in the olfactory epithelium of the mouse

Volatile odorous chemicals are detected by around a thousand different G protein-coupled odorant receptors in the mouse. We demonstrated that exposure of the behaving mouse to odorant for a few minutes led to induction of the immediate early gene c-fos for several hours in a fraction of the olfactory sensory neurones in the nasal cavity. Associated with this odorant-specific induction event was activation of extracellular-regulated kinase (ERK)1/2 that preceded increased c-fos expression. The distribution of odorant-activated neurones mimicked the scattered and spatially limited distribution of neurones expressing a single odorant receptor gene. A small change in odorant chemical structure caused a zonal shift in the spatial distribution of activated neurones, suggesting that the gene expression change resulted from specific receptor interaction. Repeated exposure to odorant or use of different concentrations did not change the pattern of c-fos induction. These results indicate that odorant-induced c-fos expression can be used to visualize odorant representations in the olfactory epithelium that reflect late cellular events regulated by adequate odorant receptor stimulation.     

Development of specific adsorbents for human tumor necrosis factor-alpha: influence of antibody immobilization on performance and biocompatibility

To develop adsorbents for the specific removal of tumor necrosis factor-alpha (TNF) in extracorporeal blood purification, cellulose microparticles were functionalized either with a monoclonal anti-TNF antibody (mAb) or with recombinant human antibody fragments (Fab). The TNF binding capacity of the adsorbents was determined with in vitro batch experiments using spiked human plasma (spike: 1200 pg TNF/mL; 1 mg particles in 250 muL plasma). Random immobilization of the full-sized monoclonal antibody to periodate-activated cellulose yielded particles with excellent adsorption capacity (258.1 +/- 48.6 pg TNF per mg adsorbent wet weight). No leaching of antibody was detectable, and the adsorbents retained their activity for at least 12 months at 4 degrees C. We found that the conditions used during immobilization of the antibody (pH, nature of the reducing agent) profoundly influenced the biocompatibility of the resulting adsorbents, especially with respect to activation of the complement system. Particles obtained by random immobilization of the monovalent Fab fragments on periodate-activated cellulose using the same conditions as for immobilization of the mAb exhibited only low adsorption capacity (44 +/- 7 pg/mg adsorbent wet weight). Oriented coupling of the Fab fragments on chelate-epoxy cellulose via a C-terminal histidine tag, however, increased the adsorption capacity to 178.3 +/- 8.6 pg TNF/mg adsorbent wet weight. Thus, in the case of small, monovalent ligands, the orientation on the carrier is critical to retain full binding activity.     

In situ FTIR ATR spectroscopic study of the interaction of immobilized human tumor necrosis factor-alpha with a monoclonal antibody in aqueous environment

By in situ FTIR ATR measurements, the antibody (AB) recognition of human tumor necrosis factor-alpha (TNFalpha) immobilized on the Ge surface of a multiple internal reflection element (MIRE) was investigated. The experiments were performed in aqueous environment in a flow-through cell. After immobilization of TNFalpha on the Ge-MIRE by direct adsorption from aqueous solution, the immobilisate reached stability after about 1 h under flow-through conditions. The remaining sites of the Ge surface were saturated by bovine serum albumin (BSA) in order to prevent unspecific binding of anti-TNFalpha AB which was then added. The obtained FTIR ATR spectra were shown to result exclusively from AB specifically interacting with TNFalpha, since the absence of immunoglobulin binding to BSA adsorbed to the Ge MIRE was verified by a reference experiment. Finally, the stability of all adsorbed protein immobilisates was monitored under flow-through conditions for 10.5 h. The TNFalpha-AB complex showed a decrease of 7.4%, whereas the BSA adsorbate remained stable. IR measurements were performed with polarized light in order to study orientational effects of the immobilized proteins. The dichroic ratios and surface concentrations of all used proteins are available after quantitative analysis of the amide II bands.     

Interaction of cytochrome c with cytochrome oxidase: two different docking scenarios

Cytochrome c is the specific and efficient electron transfer mediator between the two last redox complexes of the mitochondrial respiratory chain. Its interaction with both partner proteins, namely cytochrome c(1) (of complex III) and the hydrophilic Cu(A) domain (of subunit II of oxidase), is transient, and known to be guided mainly by electrostatic interactions, with a set of acidic residues on the presumed docking site on the Cu(A) domain surface and a complementary region of opposite charges exposed on cytochrome c. Information from recent structure determinations of oxidases from both mitochondria and bacteria, site-directed mutagenesis approaches, kinetic data obtained from the analysis of isolated soluble modules of interacting redox partners, and computational approaches have yielded new insights into the docking and electron transfer mechanisms. Here, we summarize and discuss recent results obtained from bacterial cytochrome c oxidases from both Paracoccus denitrificans, in which the primary electrostatic encounter most closely matches the mitochondrial situation, and the Thermus thermophilus ba(3) oxidase in which docking and electron transfer is predominantly based on hydrophobic interactions.     

Mutations in the gene KCNV2 encoding a voltage-gated potassium channel subunit cause "cone dystrophy with supernormal rod electroretinogram" in humans

"Cone dystrophy with supernormal rod electroretinogram (ERG)" is an autosomal recessive disorder that causes lifelong visual loss combined with a supernormal ERG response to a bright flash of light. We have linked the disorder to a 0.98-cM (1.5-Mb) region on chromosome 9p24, flanked by rs1112534 and rs1074449, using homozygosity mapping in one large consanguineous pedigree. Analysis of one gene within this region, KCNV2, showed a homozygous nonsense mutation. Mutations were also found in 17 alleles of 10 other unrelated families with the same disorder. In situ hybridization demonstrated KCNV2 expression in human rod and cone photoreceptors. The precise function of KCNV2 in human photoreceptors remains to be determined, although this work suggests that mutations might perturb or abrogate I(KX), the potassium current within vertebrate photoreceptor inner segments, which has been shown to set their resting potential and voltage response.