The long periodicity phase (LPP) controversy part I: The influence of a natural-like ratio of the CER[EOS] analogue [EOS]-br in a CER[NP]/[AP] based stratum corneum modelling system: A neutron diffraction study

This study used neutron diffraction to investigate a ceramide-[NP] C24/[AP] C24 /[EOS]-br C30/cholesterol/lignoceric acid (0.6: 0.3: 0.1: 0.7: 1) based stratum corneum modelling system. By adding specifically deuterated ceramides-[NP]-D₃, [AP]-D₃, and [EOS]-br-D₃, detailed information on the lamellar and the nanostructure of the system was obtained. For the short periodicity phase a natural-like lamellar repeat distance of 5.47?±?0.02?nm was observed, similar to the [NP]/[AP] base system without the [EOS]-br. Unlike in this system the ceramides here were slightly tilted, hinting towards a slightly less natural arrangement. Due to the deuteration it was possible to observe that the long ceramide chains were overlapping in the lamellar mid-plane. This is considered to be an important feature for the natural stratum corneum. Despite the presence of a ceramide [EOS] analogue – able to form a long phase arrangement – no distinct long periodicity phase was formed, despite a slightly higher than natural ω-acyl ceramide ratio of 10?mol%. The deuterated variant of this ceramide determined that the very long ceramide was integrated into the short periodicity phase, spanning multiple layers instead. The – compared to the base system – unchanged repeat distance highlights the stability of this structure. Furthermore, the localisation of the very long ceramide in the short periodicity phase indicates the possibility of a crosslinking effect and thus a multilayer stabilizing role for the ceramide [EOS]. It can be concluded, that additionally to the mere presence of ceramide-[EOS] more complex conditions have to be met in order to form this long phase. This has to be further investigated in the future.     

Transduction and transfection of difficult-to-transfect cells: Systematic attempts for the transfection of protozoa Leishmania

Cell-penetrating peptides (CPPs) are used to internalize different cargoes, including DNA, into live mammalian and plant cells. Despite many cells being easily transfected with this approach, other cells are rather “difficult” or “hard to transfect,” including protist cells of the genus Leishmania. Based on our previous results in successfully internalizing proteins into Leishmania tarentolae cells, we used single CPPs and three different DNA-binding proteins to form protein-like complexes with plasmids covered with CPPs. We attempted magnetofection, electroporation, and transfection using a number of commercially available detergents. While complex formation with negatively charged DNA required substantially higher amounts of CPPs than those necessary for mostly neutral proteins, the cytotoxicity of the required amounts of CPPs and auxiliaries was thoroughly studied. We found that Leishmania cells were indeed susceptible to high concentrations of some CPPs and auxiliaries, although in a different manner compared with that for mammalian cells. The lack of successful transfections implies the necessity to accept certain general limitations regarding DNA internalization into difficult-to-transfect cells. Only electroporation allowed reproducible internalization of large and rigid plasmid DNA molecules through electrically disturbed extended membrane areas, known as permeable membrane macrodomains.     

Prediction of quaternary structure by analysis of hot spot residues in protein-protein interfaces: the case of anthranilate phosphoribosyltransferases

It is an important goal of computational biology to correctly predict the association state of a protein based on its amino acid sequence and the structures of known homologues. We have pursued this goal on the example of anthranilate phosphoribosyltransferase (AnPRT), an enzyme that is involved in the biosynthesis of the amino acid tryptophan. Firstly, known crystal structures of naturally occurring homodimeric AnPRTs were analyzed using the Protein Interfaces, Surfaces, and Assemblies (PISA) service of the European Bioinformatics Institute (EBI). This led to the identification of two hydrophobic “hot spot” amino acids in the protein-protein interface that were predicted to be essential for self-association. Next, in a comprehensive multiple sequence alignment (MSA), naturally occurring AnPRT variants with hydrophilic or charged amino acids in place of hydrophobic residues in the two hot spot positions were identified. Representative variants were characterized in terms of thermal stability, enzymatic activity, and quaternary structure. We found that AnPRT variants with charged residues in both hot spot positions exist exclusively as monomers in solution. Variants with hydrophilic amino acids in one hot spot position occur in both forms, monomer and dimer. The results of the present study provide a detailed characterization of the determinants of the AnPRT monomer-dimer equilibrium and show that analysis of hot spots in combination with MSAs can be a valuable tool in prediction of protein quaternary structures.     

Changes in the auditory neuropil after deafferentation in adult grasshoppers (Schistocerca gregaria)

Nervous systems are capable of structural adjustments. Such plastic changes also occur in the auditory system of the locust Schistocerca gregaria in which a deafferentation leads to compensatory mechanisms, such as collateral sprouting of interneurons. In this study we further investigated lesion related changes in the major auditory neuropil, the median ventral association center (mVAC) of the metathoracic ganglion. The auditory sensory organ of adult locusts was unilaterally extirpated and the mVAC was histologically and immunocytochemically analyzed until 20 days postoperative. Measurements of the neuropil area in transverse sections showed a decrease in size. The putative transmitter of the afferents, acetylcholine, was investigated by acetylcholinesterase histochemistry. Comparisons of staining intensities in the intact and deafferentated mVAC indicated that the amount of acetylcholinesterase in the deafferentated mVAC decreased shortly after the operation. Both, the decreases in size of the mVAC as well as that in acetylcholinesterase histochemistry were only less than 10% compared to the controls. The immunoreactivity against the neurotransmitters γ-amino butyric acid and serotonin was not influenced by the deafferentation.     

beta-thymosin is required for axonal tract formation in developing zebrafish brain

beta-Thymosins are polypeptides that bind monomeric actin and thereby function as actin buffers in many cells. We show that during zebrafish development, &bgr;-thymosin expression is tightly correlated with neuronal growth and differentiation. It is transiently expressed in a subset of axon-extending neurons, essentially primary neurons that extend long axons, glia and muscle. Non-neuronal expression in the brain is restricted to a subset of glia surrounding newly forming axonal tracts. Skeletal muscle cells in somites, jaw and fin express beta-thymosin during differentiation, coinciding with the time of innervation. Injection of beta-thymosin antisense RNA into zebrafish embryos results in brain defects and impairment of the development of beta-thymosin-associated axon tracts. Furthermore, irregularities in somite formation can be seen in a subset of embryos. Compared to wild-type, antisense-injected embryos show slightly weaker and more diffuse engrailed staining at the midbrain-hindbrain boundary and a strong reduction of Isl-1 labeling in Rohon Beard and trigeminal neurons. The decreased expression is not based on a loss of neurons indicating that beta-thymosin may be involved in the maintenance of the expression of molecules necessary for neuronal differentiation. Taken together, our results strongly indicate that beta-thymosin is an important regulator of development.     

Placental failure and impaired vasculogenesis result in embryonic lethality for neuropathy target esterase-deficient mice

Age-dependent neurodegeneration resulting from widespread apoptosis of neurons and glia characterize the Drosophila Swiss Cheese (SWS) mutant. Neuropathy target esterase (NTE), the vertebrate homologue of SWS, reacts with organophosphates which initiate a syndrome of axonal degeneration. NTE is expressed in neurons and a variety of nonneuronal cell types in adults and fetal mice. To investigate the physiological functions of NTE, we inactivated its gene by targeted mutagenesis in embryonic stem cells. Heterozygous NTE(+/-) mice displayed a 50% reduction in NTE activity but underwent normal organ development. Complete inactivation of the NTE gene resulted in embryonic lethality, which became evident after gastrulation at embryonic day 9 postcoitum (E9). As early as E7.5, mutant embryos revealed growth retardation which did not reflect impaired cell proliferation but rather resulted from failed placental development; as a consequence, massive apoptosis within the developing embryo preceded its resorption. Histological analysis indicated that NTE is essential for the formation of the labyrinth layer and survival and differentiation of secondary giant cells. Additionally, impairment of vasculogenesis in the yolk sacs and embryos of null mutant conceptuses suggested that NTE is also required for normal blood vessel development.     

The Sp8 zinc-finger transcription factor is involved in allometric growth of the limbs in the beetle Tribolium castaneum

Members of the Sp gene family are involved in a variety of developmental processes in both vertebrates and invertebrates. We identified the ortholog of the Drosophila Sp-1 gene in the red flour beetle Tribolium castaneum, termed T-Sp8 because of its close phylogenetic relationship to the vertebrate Sp8 genes. During early embryogenesis, T-Sp8 is seen in segmental stripes. During later stages, TSp8 is dynamically expressed in the limb buds of the Tribolium embryo. At the beginning of bud formation, TSp8 is uniformly expressed in all body appendages. As the limbs elongate, a ring pattern develops sequentially and the expression profile at the end of embryogenesis correlates with the final length of the appendage. In limbs that do not grow out like the labrum and the labium, T-Sp8 expression remains uniform, whereas a two-ring pattern develops in the longer antennae and the maxillae. In the legs that elongate even further, four rings of T-Sp8 expression can be seen at the end of leg development. The role of T-Sp8 for appendage development was tested using RNAi. Upon injection of double stranded T-Sp8 RNA, larvae develop with dwarfed appendages. Affected T-Sp8(RNAi) legs were tested for the presence of medial and distal positional values using the expression marker genes dachshund and Distal-less, respectively. The results show that a dwarfed TSp8(RNAi) leg consists of proximal, medial and distal parts and argues against T-Sp8 being a leg gap gene. Based on the differential expression pattern of T-Sp8 in the appendages of the head and the thorax and the RNAi phenotype, we hypothesise that T-Sp8 is involved in the regulation of limb-length in relation to body size - a process called allometric growth.     

Developmental origin of wiring specificity in the olfactory system of Drosophila

In both insects and mammals, olfactory receptor neurons (ORNs) expressing specific olfactory receptors converge their axons onto specific glomeruli, creating a spatial map in the brain. We have previously shown that second order projection neurons (PNs) in Drosophila are prespecified by lineage and birth order to send their dendrites to one of approximately 50 glomeruli in the antennal lobe. How can a given class of ORN axons match up with a given class of PN dendrites? Here, we examine the cellular and developmental events that lead to this wiring specificity. We find that, before ORN axon arrival, PN dendrites have already created a prototypic map that resembles the adult glomerular map, by virtue of their selective dendritic localization. Positional cues that create this prototypic dendritic map do not appear to be either from the residual larval olfactory system or from glial processes within the antennal lobe. We propose instead that this prototypic map might originate from both patterning information external to the developing antennal lobe and interactions among PN dendrites.