D-ribose utilisation differentiates porcine Brachyspira pilosicoli from other porcine Brachyspira species

D-ribose utilisation was studied in 60 Brachyspira pilosicoli strains and 35 strains of other Brachyspira species, the majority of which were of porcine origin. Utilisation of D-ribose was demonstrated indirectly by measuring the reduction in pH of densely inoculated tryptone-peptone broth supplemented with 7% foetal calf serum and 1% D-ribose. Among B. pilosicoli strains, the mean reduction in pH units was 1.72 (range 0.95-2.28) in broth with D-ribose and 0.27 (range 0.10-0.40) in sugar-free control broth. For Brachyspira strains other than B. pilosicoli, the corresponding reductions in pH units were 0.37 (range 0.12-0.49) and 0.37 (range 0.15-0.58). In conclusion, porcine B. pilosicoli can be differentiated from other porcine Brachyspira species by a test for D-ribose utilisation.     

Structure and dynamics of sphingomyelin bilayer: insight gained through systematic comparison to phosphatidylcholine

Sphingomyelin, one of the main lipid components of biological membranes, is actively involved in various cellular processes such as protein trafficking and signal transduction. In particular, specific lateral domains enriched in sphingomyelin and cholesterol have been proposed to play an important functional role in biomembranes, although their precise characteristics have remained unclear. A thorough understanding of the functional role of membranes requires detailed knowledge of their individual lipid components. Here, we employ molecular dynamics simulations to conduct a systematic comparison of a palmitoylsphingomyelin (PSM, 16:0-SM) bilayer with a membrane that comprises dipalmitoylphosphatidylcholine (DPPC) above the main phase transition temperature. We clarify atomic-scale properties that are specific to sphingomyelin due to its sphingosine moiety, and further discuss their implications for SM-rich membranes. We find that PSM bilayers, and in particular the dynamics of PSM systems, are distinctly different from those of a DPPC bilayer. When compared with DPPC, the strong hydrogen bonding properties characteristic to PSM are observed to lead to considerable structural changes in the polar headgroup and interface regions. The strong ordering of PSM acyl chains and specific ordering effects in the vicinity of a PSM-water interface reflect this issue clearly. The sphingosine moiety and related hydrogen bonding further play a crucial role in the dynamics of PSM bilayers, as most dynamic properties, such as lateral and rotational diffusion, are strongly suppressed. This is most evident in the rotational motion characterized by spin-lattice relaxation times and the decay of hydrogen bond autocorrelation functions that are expected to be important in complexation of SM with other lipids in many-component bilayers. A thorough understanding of SM bilayers would greatly benefit from nuclear magnetic resonance experiments for acyl chain ordering and dynamics, allowing full comparison of these simulations to experiments.     

Stimulation of ectodermal organ development by Ectodysplasin-A1

Organs developing as ectodermal appendages share similar early morphogenesis and molecular mechanisms. Ectodysplasin, a signaling molecule belonging to the tumor necrosis factor family, and its receptor Edar are required for normal development of several ectodermal organs in humans and mice. We have overexpressed two splice forms of ectodysplasin, Eda-A1 and Eda-A2, binding to Edar and another TNF receptor, Xedar, respectively, under the keratin 14 (K14) promoter in the ectoderm of transgenic mice. Eda-A2 overexpression did not cause a detectable phenotype. On the contrary, overexpression of Eda-A1 resulted in alterations in a variety of ectodermal organs, most notably in extra organs. Hair development was initiated continuously from E14 until birth, and in addition, the transgenic mice had supernumerary teeth and mammary glands, phenotypes not reported previously in transgenic mice. Also, hair composition and structure was abnormal, and the cycling of hairs was altered so that the growth phase (anagen) was prolonged. Both hairs and nails grew longer than normal. Molar teeth were of abnormal shape, and enamel formation was severely disturbed in incisors. Furthermore, sweat gland function was stimulated and sebaceous glands were enlarged. We conclude that ectodysplasin-Edar signaling has several roles in ectodermal organ development controlling their initiation, as well as morphogenesis and differentiation.     

In vivo tumor growth inhibition and biodistribution studies of locked nucleic acid (LNA) antisense oligonucleotides

Locked nucleic acids (LNA) are novel high-affinity DNA analogs that can be used as genotype-specific drugs. The LNA oligonucleotides (LNA PO ODNs) are very stable in vitro and in vivo without the need for a phosphorothiolated backbone. In this study we tested the biological fate and the efficacy in tumor growth inhibition of antisense oligonucleotides directed against the gene of the large subunit of RNA polymerase II (POLR2A) that are completely synthesized as LNA containing diester backbones. These full LNA oligonucleotides strongly reduce POLR2A protein levels. Full LNA PO ODNs appeared to be very stable compounds when injected into the circulation of mice. Full LNA PO ODNs were continuously administered for 14 days to tumor-bearing nude mice. Tumor growth was inhibited sequence specifically at dosages from 1 mg/kg/day. LNA PO ODNs appeared to be non-toxic at dosages <5 mg/kg/day. Biodistribution studies showed the kidneys to have the highest uptake of LNA PO ODNs and urinary secretion as the major route of clearance. This report shows that LNA PO ODNs are potent genotype-specific drugs that can inhibit tumor growth in vivo.     

S-adenosylmethionine radical enzymes

The role of S-adenosylmethionine (SAM) as a precursor to organic radicals, generated by one-electron reduction of SAM and subsequent fission to form 5'-deoxyadenosyl radical and methionine, has been known for some time. Only recently, however, has it become apparent how widespread such enzymes are, and what a wide range of chemical reactions they catalyze. In the last few years several new SAM radical enzymes have been identified. Spectroscopic and kinetic investigations have begun to uncover the mechanism by which an iron sulfur cluster unique to these enzymes reduces SAM to generate adenosyl radical. Most recently, the first X-ray structures of SAM radical enzymes, coproporphyrinogen-III oxidase, and biotin synthase have been solved, providing a structural framework within which to interpret mechanistic studies.     

A novel specific role for I kappa B kinase complex-associated protein in cytosolic stress signaling

We demonstrate here a novel role for the I kappa B kinase complex-associated protein (IKAP) in the regulation of activation of the mammalian stress response via the c-Jun N-terminal kinase (JNK)-signaling pathway. We cloned IKAP as a JNK-associating protein using the Ras recruitment yeast two-hybrid system. IKAP efficiently and specifically enhanced JNK activation induced by ectopic expression of MEKK1 and ASK1, upstream activators of JNK. Importantly, IKAP also enhanced JNK activation induced by ultraviolet light irradiation as well as treatments with tumor necrosis factor or epidermal growth factor. The JNK association site in IKAP was mapped to the C-terminal part of IKAP. Interestingly, this region is deleted from IKAP expressed in the autonomous nervous system of the patients affected by familial dysautonomia. Ectopic expression of this C-terminal fragment of IKAP was sufficient to support JNK activation. Taken together, our data demonstrate a novel role for IKAP in the regulation of the JNK-mediated stress signaling. Additionally, our results point to a role of JNK signaling in familial dysautonomia and, thus, further support the involvement of JNK signaling in the development, survival, and degeneration of the sensory and autonomic nervous system.     

Selective protein exit from yeast endoplasmic reticulum in absence of functional COPII coat component Sec13p

Sec13p has been thought to be an essential component of the COPII coat, required for exit of proteins from the yeast endoplasmic reticulum (ER). We show herein that normal function of Sec13p was not required for ER exit of the Hsp150 glycoprotein. Hsp150 was secreted to the medium under restrictive conditions in a sec13-1 mutant. The COPII components Sec23p and Sec31p and the GTP/GDP exchange factor Sec12p were required in functional form for secretion of Hsp150. Hsp150 leaves the ER in the absence of retrograde COPI traffic, and the responsible determinant is a peptide repeated 11 times in the middle of the Hsp150 sequence. Herein, we localized the sorting determinant for Sec13p-independent ER exit to the C-terminal domain. Sec13p-dependent invertase left the ER in the absence of normal Sec13p function, when fused to the C-terminal domain of Hsp150, demonstrating that this domain contained an active mediator of Sec13p-independent secretion. Thus, Hsp150 harbors two different signatures that regulate its ER exit. Our data show that transport vesicles lacking functional Sec13p can carry out ER-to-Golgi transport, but select only specific cargo protein(s) for ER exit.     

Regulation of hair follicle development by the TNF signal ectodysplasin and its receptor Edar

X-linked and autosomal forms of anhidrotic ectodermal dysplasia syndromes (HED) are characterized by deficient development of several ectodermal organs, including hair, teeth and exocrine glands. The recent cloning of the genes that underlie these syndromes, ectodysplasin (ED1) and the ectodysplasin A receptor (EDAR), and their identification as a novel TNF ligand-receptor pair suggested a role for TNF signaling in embryonic morphogenesis. In the mouse, the genes of the spontaneous mutations Tabby (Ta) and downless (dl) were identified as homologs of ED1 and EDAR, respectively. To gain insight into the function of this signaling pathway in development of skin and hair follicles, we analyzed the expression and regulation of Eda and Edar in wild type as well as Tabby and Lef1 mutant mouse embryos. We show that Eda and Edar expression is confined to the ectoderm and occurs in a pattern that suggests a role of ectodysplasin/Edar signaling in the interactions between the ectodermal compartments and the formation and function of hair placodes. By using skin explant cultures, we further show that this signaling pathway is intimately associated with interactions between the epithelial and mesenchymal tissues. We also find that Ta mutants lack completely the placodes of the first developing tylotrich hairs, and that they do not show patterned expression of placodal genes, including Bmp4, Lef1, Shh, Ptch and Edar, and the genes for beta-catenin and activin A. Finally, we identified activin as a mesenchymal signal that stimulates Edar expression and WNT as a signal that induces Eda expression, suggesting a hierarchy of distinct signaling pathways in the development of skin and hair follicles. In conclusion, we suggest that Eda and Edar are associated with the onset of ectodermal patterning and that ectodysplasin/edar signaling also regulates the morphogenesis of hair follicles.     

Photoinhibition and loss of photosystem II reaction centre proteins during senescence of soybean leaves. Enhancement of photoinhibition by the 'stay-green' mutation cytG

The 'stay-green' mutation cytG in soybean ( Glycine max ) partially inhibits the degradation of the light-harvesting complex II (LHCII) and the associated chlorophyll during monocarpic senescence. cytG did not alter the breakdown of the cytochrome b 6/ f complex, thylakoid ATP synthase or components of Photosystem I. In contrast, cytG accelerated the loss of oxygen evolution activity and PSII reaction-centre proteins. These data suggest that LHCII and other thylakoid components are degraded by separate pathways. In leaves induced to senesce by darkness, cytG inhibited the breakdown of LHCII and chlorophyll, but it did not enhance the loss of PSII-core components, indicating that the accelerated degradation of PSII reaction centre proteins in cytG was light dependent. Illumination of mature and senescent leaves of wild-type soybean in the presence of an inhibitor (lincomycin) of chloroplast protein synthesis revealed that senescence per se did not affect the rate of photoinhibition in leaves. Likewise, mature leaves of the cytG mutant did not show more photoinhibition than wild-type leaves. However, in senescent cytG leaves, photoinhibition proceeded more rapidly than in the wild-type. We conclude that the cytG mutation enhances photoinhibition in senescing leaves, and photoinhibition causes the rapid loss of PSII reaction-centre proteins during senescence in cytG .     

Development of Baltic Sea zooplankton in the presence of a toxic cyanobacterium: a mesocosm approach

Cyanobacteria blooms are common in the Baltic Sea and are consideredto be a poor food source and sometimes toxic to zooplankton.Most experiments demonstrating harmful effects have been short-termincubations with monocultures or simple mixtures of food. Inthis study, a mesocosm approach was used to examine zooplanktonresponses over generation timescales. A toxic strain of thecyanobacterium Nodularia spumigena was added to bag enclosuresof ambient water. The initial mesozooplankton concentrationwas either reduced by prescreening the water or enriched withlocally caught zooplankton. Experiments ran for 15 days, longenough to monitor reproductive success and development of thenext mesozooplankton generations. There was no major harmfuleffect on the zooplankton assemblage, even though the concentrationof the toxin nodularin was in the upper range of field observations.The copepod Eurytemora affinis, rotifers Synchaeta spp. andthe cladoceran Bosmina longispina maritima were able to developand reproduce successfully in the presence of N. spumigena.The only species showing impaired recruitment was the copepodAcartia bifilosa. The general lack of population level effectsfrom N. spumigena in this study can be reconciled with previousobservations of adverse effects. Cyanobacteria alone may bepoor food and toxic to zooplankton, but in the mesocosms a richassemblage of microbiota developed, similar to that associatedwith blooms in the field. We suggest that, in the context ofotherwise food-depleted summer situations in the open BalticSea, zooplankton can derive benefit from cyanobacteria bloomassemblages.