Physicochemical and MRI characterization of Gd<Superscript>3+</Superscript>-loaded polyamidoamine and hyperbranched dendrimers

Generation 4 polyamidoamine (PAMAM) and, for the first time, hyperbranched poly(ethylene imine) or polyglycerol dendrimers have been loaded with Gd³⁺ chelates, and the macromolecular adducts have been studied in vitro and in vivo with regard to MRI contrast agent applications. The Gd³⁺ chelator was either a tetraazatetracarboxylate DOTA-pBn⁴⁻ or a tetraazatricarboxylate monoamide DO3A-MA³⁻ unit. The water exchange rate was determined from a ¹⁷O NMR and ¹H Nuclear Magnetic Relaxation Dispersion study for the corresponding monomer analogues [Gd(DO3A-AEM)(H₂O)] and [Gd(DOTA-pBn-NH₂)(H₂O)]⁻ (k _ex²⁹⁸ = 3.4 and 6.6 × 10⁶ s⁻¹, respectively), where H₃DO3A-AEM is {4-[(2-acetylaminoethylcarbamoyl)methyl]-7,10-bis(carboxymethyl-1,4,7,10-tetraazacyclododec-1-yl)}-acetic acid and H₄DOTA-pBn-NH₂ is 2-(4-aminobenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid. For the macromolecular complexes, variable-field proton relaxivities have been measured and analyzed in terms of local and global motional dynamics by using the Lipari–Szabo approach. At frequencies below 100 MHz, the proton relaxivities are twice as high for the dendrimers loaded with the negatively charged Gd(DOTA-pBn)⁻ in comparison with the analogous molecule bearing the neutral Gd(DO3A-MA). We explained this difference by the different rotational dynamics: the much slower motion of Gd(DOTA-pBn)⁻-loaded dendrimers is likely related to the negative charge of the chelate which creates more rigidity and increases the overall size of the macromolecule compared with dendrimers loaded with the neutral Gd(DO3A-MA). Attachment of poly(ethylene glycol) chains to the dendrimers does not influence relaxivity. Both hyperbranched structures were found to be as good scaffolds as regular PAMAM dendrimers in terms of the proton relaxivity of the Gd³⁺ complexes. The in vivo MRI studies on tumor-bearing mice at 4.7 T proved that all dendrimeric complexes are suitable for angiography and for the study of vasculature parameters like blood volume and permeability of tumor vessels. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Free-air exposure systems to scale up ozone research to mature trees

Because seedlings and mature trees do not necessarily respond similarly to O(3) stress, it is critically important that exposure systems be developed that allow exposure of seedlings through to mature trees. Here we describe three different O(3) Free-Air Exposure Systems that have been used successfully for exposure at all growth stages. These systems of spatially uniform O(3) release have been shown to provide reliable O(3) exposure with minimal, if any, impact on the microclimate. This methodology offers a welcome alternative to chamber studies which had severe space constraints precluding stand or community-level studies and substantial chamber effects on the microclimate and, hence physiological tree performance.     

Synopsis of the CASIROZ case study: carbon sink strength of Fagus sylvatica L. in a changing environment--experimental risk assessment of mitigation by chronic ozone impact

Databases are needed for the ozone (O(3)) risk assessment on adult forest trees under stand conditions, as mostly juvenile trees have been studied in chamber experiments. A synopsis is presented here from an integrated case study which was conducted on adult FAGUS SYLVATICA trees at a Central-European forest site. Employed was a novel free-air canopy O(3) fumigation methodology which ensured a whole-plant assessment of O(3) sensitivity of the about 30 m tall and 60 years old trees, comparing responses to an experimental 2 x ambient O(3) regime (2 x O(3), max. 150 nl O(3) l (-1)) with those to the unchanged 1 x ambient O(3) regime (1 x O(3)=control) prevailing at the site. Additional experimentation on individual branches and juvenile beech trees exposed within the forest canopy allowed for evaluating the representativeness of young-tree and branch-bag approaches relative to the O(3) sensitivity of the adult trees. The 2 x O(3) regime did not substantially weaken the carbon sink strength of the adult beech trees, given the absence of a statistically significant decline in annual stem growth; a 3 % reduction across five years was demonstrated, however, through modelling upon parameterization with the elaborated database. 2 x O(3) did induce a number of statistically significant tree responses at the cell and leaf level, although the O(3) responsiveness varied between years. Shade leaves displayed an O(3) sensitivity similar to that of sun leaves, while indirect belowground O(3) effects, apparently mediated through hormonal relationships, were reflected by stimulated fine-root and ectomycorrhizal development. Juvenile trees were not reliable surrogates of adult ones in view of O(3) risk assessment. Branch sections enclosed in (climatized) cuvettes, however, turned out to represent the O(3) sensitivity of entire tree crowns. Drought-induced stomatal closure decoupled O(3) intake from O(3) exposure, as in addition, also the "physiologically effective O(3) dose" was subject to change. No evidence emerged for a need to lower the "Critical Level for Ozone" in risk assessment of forest trees, although sensitive tree parameters did not necessarily reflect a linear relationship to O(3) stress. Exposure-based concepts tended to overestimate O(3) risk under drought, which is in support of current efforts to establish flux-related concepts of O(3) intake in risk assessment.     

Characterization of cysteine-degrading and H2S-releasing enzymes of higher plants - from the field to the test tube and back

Due to the clean air acts and subsequent reduction of emission of gaseous sulfur compounds sulfur deficiency became one of the major nutrient disorders in Northern Europe. Typical sulfur deficiency symptoms can be diagnosed. Especially plants of the Cruciferae family are more susceptible against pathogen attack. Sulfur fertilization can in part recover or even increase resistance against pathogens in comparison to sulfur-deficient plants. The term sulfur-induced resistance (SIR) was introduced, however, the molecular basis for SIR is largely unknown. There are several sulfur-containing compounds in plants which might be involved in SIR, such as high levels of thiols, glucosinolates, cysteine-rich proteins, phytoalexins, elemental sulfur, or H2S. Probably more than one strategy is used by plants. Species- or even variety-dependent differences in the development of SIR are probably used. Our research focussed mainly on the release of H2S as defence strategy. In field experiments using different BRASSICA NAPUS genotypes it was shown that the genetic differences among BRASSICA genotypes lead to differences in sulfur content and L-cysteine desulfhydrase activity. Another field experiment demonstrated that sulfur supply and infection with PYRENOPEZIZA BRASSICA influenced L-cysteine desulfhydrase activity in BRASSICA NAPUS. Cysteine-degrading enzymes such as cysteine desulfhydrases are hypothesized to be involved in H2S release. Several L- and D-cysteine-specific desulfhydrase candidates have been isolated and partially analyzed from the model plant ARABIDOPSIS THALIANA. However, it cannot be excluded that H2S is also released in a partial back reaction of O-acetyl-L-serine(thiol)lyase or enzymes not yet characterized. For the exact determination of the H2S concentration in the cell a H2S-specific microsensor was used the first time for plant cells. The transfer of the results obtained for application back on BRASSICA was initiated.     

Molecular mechanisms underlying the probiotic effects of Escherichia coli Nissle 1917 involve ZO-2 and PKCzeta redistribution resulting in tight junction and epithelial barrier repair

The probiotic Escherichia coli strain Nissle 1917 (EcN) has been used for decades in human medicine in Central Europe for the treatment and prevention of intestinal disorders and diseases. However, the molecular mechanisms underlying its beneficial effects are only partially understood. To identify molecular responses induced by EcN that might contribute to its probiotic properties polarized T84 cells were investigated employing DNA microarrays, quantitative RT-PCR, Western blotting, immunofluorescence and specific protein kinase C (PKC) inhibitors. Polarized T84 epithelial cell monolayers were used as a model to monitor barrier disruption by infection with the enteropathogenic E. coli (EPEC) strain E2348/69. Co-incubation of EPEC with EcN or addition of EcN following EPEC infection abolished barrier disruption and, moreover, restored barrier integrity as monitored by transepithelial resistance. DNA-microarray analysis of T84 cells incubated with EcN identified 300+ genes exhibiting altered expression. EcN altered the expression, distribution of zonula occludens-2 (ZO-2) protein and of distinct PKC isotypes. ZO-2 expression was enhanced in parallel to its redistribution towards the cell boundaries. This study provides evidence that EcN induces an overriding signalling effect leading to restoration of a disrupted epithelial barrier. This is transmitted via silencing of PKCzeta and the redistribution of ZO-2. We suggest that these properties contribute to the reported efficacy in the treatment of inflammatory bowel diseases and in part rationalize the probiotic nature of EcN.     

Mutation in the Scyl1 gene encoding amino-terminal kinase-like protein causes a recessive form of spinocerebellar neurodegeneration

Here, we show that the murine neurodegenerative disease mdf (autosomal recessive mouse mutant 'muscle deficient') is caused by a loss-of-function mutation in Scyl1, disrupting the expression of N-terminal kinase-like protein, an evolutionarily conserved putative component of the nucleocytoplasmic transport machinery. Scyl1 is prominently expressed in neurons, and enriched at central nervous system synapses and neuromuscular junctions. We show that the pathology of mdf comprises cerebellar atrophy, Purkinje cell loss and optic nerve atrophy, and therefore defines a new animal model for neurodegenerative diseases with cerebellar involvement in humans.     

HAP1 can sequester a subset of TBP in cytoplasmic inclusions via specific interaction with the conserved TBP<Subscript>CORE</Subscript>

Background  

Huntington's disease, spinal and bulbar muscular atrophy, and spinocerebellar ataxia 17 (SCA17) are caused by expansions in the polyglutamine (polyQ) repeats in Huntingtin protein (Htt), androgen receptor protein (AR), and TATA-binding protein (TBP), respectively. Htt-associated protein 1 (HAP1), a component of neuronal cytoplasmic stigmoid bodies (STBs), can sequester polyQ-expanded Htt and AR in STBs, thereby antagonizing formation of the nuclear aggregates associated with apoptotic neuron loss and disease progression.     

Generation of mice deficient for Lbx2, a gene expressed in the urogenital system, nervous system, and Pax3 dependent tissues

Lbx2 is a member of the ladybird family of homeobox genes. The first murine ortholog identified, Lbx1, is required for hypaxial musculature and dorsal spinal cord neuron development. The second murine ortholog, Lbx2, is expressed in the developing urogenital and nervous systems. To elucidate the function of Lbx2, we generated a gene-targeted allele of Lbx2 in mice. Lbx2 deficiency did not impair mouse development, and Lbx2 null mice appeared healthy and fertile. Replacement of Lbx2 by the lacZ gene provides a valuable histological marker for Lbx2-expressing cells. Given the important role of Pax3 in neural crest, we intercrossed our Lbx2 deficient mice with Splotch Pax3 mutant mice to determine if Pax3 affects Lbx2 expression. There was reduced Lbx2 expression in dorsal root ganglia and cranial nerve ganglia with Pax3 deficiency, but not in the genital tubercle. This suggested that Pax3 is required for Lbx2 expression in affected neural crest-derived tissues.