Effect of fire on the topsoil seed banks of rehabilitated bauxite mine sites in the jarrah forest of Western Australia

Summary Germinable seed stores of 5- and 8-year-old rehabilitated bauxite mine pits in south-west Western Australia were assessed before and after burning. These seed stores were compared to those of adjacent unmined Jarrah ( Eucalyptus marginata ) forest, to identify at what age fire can be reintroduced, in order to measure restoration success and reduce fire hazard. Soils were sampled in early summer (before fire) and late autumn (after fire). Before fire, the mean topsoil seed bank of 5-year-old sites was 2121 seeds per m² while 8-year-old sites had a mean of 1520 seeds per m². Only the 5-year-old sites were significantly different from the forest mean of 1478 seeds per m² for the same season. After summer burns (and possibly due to seasonal effects) topsoil seed banks of rehabilitated areas (sampled in autumn) decreased by an average of 53 per cent. Topsoil seed banks of 5–8-year-old sites were resistant to lower intensity burns, with 362 seeds per m² of native species surviving mild burns and 108 seeds per m² of native species surviving after an intense summer fire. The topsoil seed reserve of 5–8-year-old rehabilitated areas had a high proportion of annual weed species while the forest sites had high levels of subshrubs and native annuals. Low-intensity burns did not alter the composition of life-forms in the soil seed bank, while intense burns favoured annual weed and shrub species. The results indicate that it is not appropriate to introduce fire to rehabilitated areas before 8 years, due to limited fuel reduction benefits and possible adverse effects on obligate seeding species. The large proportion of weed species in the soil seed bank of young rehabilitated areas is a concern, and remains a major consideration for future disturbance of these areas.     

Evidence that NF-κB and MAPK Signaling Promotes NLRP Inflammasome Activation in Neurons Following Ischemic Stroke

Multi-protein complexes, termed “inflammasomes,” are known to contribute to neuronal cell death and brain injury following ischemic stroke. Ischemic stroke increases the expression and activation of nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) Pyrin domain containing 1 and 3 (NLRP1 and NLRP3) inflammasome proteins and both interleukin (IL)-1β and IL-18 in neurons. In this study, we provide evidence that activation of either the NF-κB and MAPK signaling pathways was partly responsible for inducing the expression and activation of NLRP1 and NLRP3 inflammasome proteins and that these effects can be attenuated using pharmacological inhibitors of these two pathways in neurons and brain tissue under in vitro and in vivo ischemic conditions, respectively. Moreover, these findings provided supporting evidence that treatment with intravenous immunoglobulin (IVIg) preparation can reduce activation of the NF-κB and MAPK signaling pathways resulting in decreased expression and activation of NLRP1 and NLRP3 inflammasomes, as well as increasing expression of anti-apoptotic proteins, Bcl-2 and Bcl-xL, in primary cortical neurons and/or cerebral tissue under in vitro and in vivo ischemic conditions. In summary, these results provide compelling evidence that both the NF-κB and MAPK signaling pathways play a pivotal role in regulating the expression and activation of NLRP1 and NLRP3 inflammasomes in primary cortical neurons and brain tissue under ischemic conditions. In addition, treatment with IVIg preparation decreased the activation of the NF-κB and MAPK signaling pathways, and thus attenuated the expression and activation of NLRP1 and NLRP3 inflammasomes in primary cortical neurons under ischemic conditions. Hence, these findings suggest that therapeutic interventions that target inflammasome activation in neurons may provide new opportunities in the future treatment of ischemic stroke.     

Maternal X chromosome expression in mouse chorionic ectoderm

An electrophoretic variant of the X-linked enzyme phosphoglycerate kinase (PGK-1) has been used to study regulation of X chromosome expression in the diploid derivatives of the trophectoderm at 8–8.5 days post coitum in the mouse. These derivatives included the chorionic ectoderm and the polar trophoblast. The biochemical analysis suggests that only the maternally derived X chromosome (X^m) is expressed in the diploid trophectoderm derivatives. Cell selection and maternal tissue contamination were ruled out as possible causes of the observed X^m expression. From these and other results, we conclude that all derivatives of the trophectoderm, along with the primitive endoderm, express only X^m, whereas derivatives of the primitive ectoderm show random X chromosome expression.     

Conserved Glu40 and Glu433 of the biotin carboxylase domain of yeast pyruvate carboxylase I isoenzyme are essential for the association of tetramers

The native form of pyruvate carboxylase is an alpha4 tetramer but the tetramerisation domain of each subunit is currently unknown. To identify this domain we co-expressed yeast pyruvate carboxylase 1 isozyme (Pyc1) with an N-terminal myc tag, together with constructs encoding either the biotin carboxylase (BC) domain or the transcarboxylase-biotin carboxyl carrier domain (TC-BCC), each with an N-terminal 9-histidine tag. From tag-affinity chromatography experiments, the subunit contacts within the tetramer were identified to be primarily located in the 55 kDa BC domain. From modelling studies based on known structures of biotin carboxylase domains and subunits we have predicted that Arg36 and Glu433 and Glu40 and Lys426, respectively, are involved pairwise in subunit interactions and are located on opposing subunits in the putative subunit interface of Pyc1. Co-expression of mutant forms with wild type Pyc1 showed that the R36E mutation had no effect on the interaction of these subunits with those of wild type Pyc1, while the E40R, E433R and R36E:E433R mutations caused severe loss of interaction with wild type Pyc1. Ultracentrifugal analysis of these mutants when expressed and purified separately indicated that the predominant form of E40R, E433R and R36R:E433R mutants is the monomer, and that their specific activities are less than 2% of the wild type. Studies on the association state and specific activity of the R36E mutant at different concentrations showed it to be much more susceptible to tetramer dissociation and inactivation than the wild type. Our results suggest that Glu40 and Glu433 play essential roles in subunit interactions.     

Assessing the significance of conserved genomic aberrations using high resolution genomic microarrays

Genomic aberrations recurrent in a particular cancer type can be important prognostic markers for tumor progression. Typically in early tumorigenesis, cells incur a breakdown of the DNA replication machinery that results in an accumulation of genomic aberrations in the form of duplications, deletions, translocations, and other genomic alterations. Microarray methods allow for finer mapping of these aberrations than has previously been possible; however, data processing and analysis methods have not taken full advantage of this higher resolution. Attention has primarily been given to analysis on the single sample level, where multiple adjacent probes are necessarily used as replicates for the local region containing their target sequences. However, regions of concordant aberration can be short enough to be detected by only one, or very few, array elements. We describe a method called Multiple Sample Analysis for assessing the significance of concordant genomic aberrations across multiple experiments that does not require a-priori definition of aberration calls for each sample. If there are multiple samples, representing a class, then by exploiting the replication across samples our method can detect concordant aberrations at much higher resolution than can be derived from current single sample approaches. Additionally, this method provides a meaningful approach to addressing population-based questions such as determining important regions for a cancer subtype of interest or determining regions of copy number variation in a population. Multiple Sample Analysis also provides single sample aberration calls in the locations of significant concordance, producing high resolution calls per sample, in concordant regions. The approach is demonstrated on a dataset representing a challenging but important resource: breast tumors that have been formalin-fixed, paraffin-embedded, archived, and subsequently UV-laser capture microdissected and hybridized to two-channel BAC arrays using an amplification protocol. We demonstrate the accurate detection on simulated data, and on real datasets involving known regions of aberration within subtypes of breast cancer at a resolution consistent with that of the array. Similarly, we apply our method to previously published datasets, including a 250K SNP array, and verify known results as well as detect novel regions of concordant aberration. The algorithm has been fully implemented and tested and is freely available as a Java application at http://www.cbil.upenn.edu/MSA.     

Localization of the hemagglutinating activity of platelet thrombospondin to a 140 000-dalton thermolytic fragment

The platelet protein thrombospondin (TSP) which is secreted from alpha-granules upon platelet activation agglutinates trypsinized, glutaraldehyde-fixed human erythrocytes. Optimal conditions for the hemagglutinating activity require that both Ca2+ and Mg2+ be present in final concentrations of 2 mM. In the presence of dithiothreitol (i.e., reduction of disulfide bonds), the lectin-like activity decreases in a manner proportional to the extent of reduction of the molecule from its native trimeric configuration into its Mr 180 000 subunits. Proteolysis of purified TSP with thermolysin, which produces discrete domains with the capacity to bind fibrinogen and heparin, also diminishes, but does not abolish, the hemagglutinating activity. Fibrinogen was without effect on hemagglutinating activity while heparin was found to be a potent inhibitor. Other proteoglycans such as hyaluronic acid, chondroitin sulfate, keratan sulfate, dermatan sulfate, and heparan sulfate had no effect. That portion of the TSP molecule apparently responsible for the hemagglutinating activity was identified by incubating a thermolytic digest of TSP with red blood cells and then determining which fragment was bound to the cell surface. The binding site resides within a peptide fragment of 140 000 daltons but is absent from an Mr 120 000 fragment derived from the Mr 140 000 fragment. Under the conditions for optimal expression of hemagglutinating activity (i.e., 2 mM MgCl2 and 2 mM CaCl2), this Mr 140 000 fragment was also shown to have heparin binding activity.