Sixteen hundred years of increasing tree cover prior to modern deforestation in Southern Amazon and Central Brazilian savannas

Tropical ecosystems are under increasing pressure from land‐use change and deforestation. Changes in tropical forest cover are expected to affect carbon and water cycling with important implications for climatic stability at global scales. A major roadblock for predicting how tropical deforestation affects climate is the lack of baseline conditions (i.e., prior to human disturbance) of forest–savanna dynamics. To address this limitation, we developed a long‐term analysis of forest and savanna distribution across the Amazon–Cerrado transition of central Brazil. We used soil organic carbon isotope ratios as a proxy for changes in woody vegetation cover over time in response to fluctuations in precipitation inferred from speleothem oxygen and strontium stable isotope records. Based on stable isotope signatures and radiocarbon activity of organic matter in soil profiles, we quantified the magnitude and direction of changes in forest and savanna ecosystem cover. Using changes in tree cover measured in 83 different locations for forests and savannas, we developed interpolation maps to assess the coherence of regional changes in vegetation. Our analysis reveals a broad pattern of woody vegetation expansion into savannas and densification within forests and savannas for at least the past ~1,600 years. The rates of vegetation change varied significantly among sampling locations possibly due to variation in local environmental factors that constrain primary productivity. The few instances in which tree cover declined (7.7% of all sampled profiles) were associated with savannas under dry conditions. Our results suggest a regional increase in moisture and expansion of woody vegetation prior to modern deforestation, which could help inform conservation and management efforts for climate change mitigation. We discuss the possible mechanisms driving forest expansion and densification of savannas directly (i.e., increasing precipitation) and indirectly (e.g., decreasing disturbance) and suggest future research directions that have the potential to improve climate and ecosystem models.     

Wnts as essential growth factors for the adult small intestine and colon

The study of physiologic functions of Wnt proteins has been complicated by the redundant nature of the families encoding the Wnt factors and their Frizzled receptors. Adenoviral expression of the secreted Wnt antagonist Dickkopf-1 (Dkk1) was used to achieve fully conditional inhibition of canonical Wnt signaling in adult mice. Systemic expression of Dkk1 resulted in rapid inhibition of Wnt target gene expression and of proliferation of the small intestine and colon, loss of proliferative crypts, and eventual inflammation and architectural degeneration. These studies indicate an essential requirement for extracellular Wnt signaling in the maintenance of adult small intestine and colon proliferation. The essential role of Wnt signaling in ongoing proliferation in the colon suggests potential clinical applications in mucosal repair for inflammatory bowel diseases and underscores the utility of adenoviral strategies for conditional ablation of gene function in adult organisms.     

X-linked cone-rod dystrophy (locus COD1): identification of mutations in RPGR exon ORF15

X-linked cone-rod dystrophy (COD1) is a retinal disease that primarily affects the cone photoreceptors; the disease was originally mapped to a limited region of Xp11.4. We evaluated the three families from our original study with new markers and clinically reassessed all key recombinants; we determined that the critical intervals in families 2 and 3 overlapped the RP3 locus and that a status change (from affected to probably unaffected) of a key recombinant individual in family 1 also reassigned the disease locus to include RP3 as well. Mutation analysis of the entire RPGR coding region identified two different 2-nucleotide (nt) deletions in ORF15, in family 2 (delAG) and in families 1 and 3 (delGG), both of which result in a frameshift leading to altered amino acid structure and early termination. In addition, an independent individual with X-linked cone-rod dystrophy demonstrated a 1-nt insertion (insA) in ORF15. The presence of three distinct mutations associated with the same disease phenotype provides strong evidence that mutations in RPGR exon ORF15 are responsible for COD1. Genetic heterogeneity was observed in three other families, including the identification of an in-frame 12-nt deletion polymorphism in ORF15 that did not segregate with the disease in one of these families.     

Novel approach to obtain biologically active recombinant heterodimeric proteins in Escherichia coli

The strategy described in this paper provides a novel approach for recombinant expression of heterodimeric proteins, and is especially suitable for the production of proteins whose characteristics lead to aggregation in E. coli expression systems. Pheromaxein, a steroid-binding protein isolated from boar saliva, is a heterodimeric protein consisting of 10x10(3) rel. mol. mass units (pheromaxein A) and 8x10(3) rel. mol. mass units (pheromaxein C) subunits. Expression of pheromaxein subunits in E. coli resulted in extensive insoluble aggregation. The difficulty faced in obtaining soluble recombinant pheromaxein subunits was clearly evident when native pheromaxein immediately formed aggregates when it was separated into its individual subunits. An increase in soluble pheromaxein expression in E. coli was obtained when the subunits were expressed as fusion proteins with thioredoxin. Pheromaxein genes were inserted separately into pET32a+ vectors at the NcoI site, resulting in thioredoxin, S.Tag and His.Tag coding regions being upstream of the inserted gene. Soluble pheromaxein A-thioredoxin (pheroA/trx) and pheromaxein C-thioredoxin (pheroC/trx) fusions were purified to homogeneity, using a laboratory scale S-protein agarose affinity column. Cleavage of thioredoxin under normal conditions was not feasible due to the extensive aggregation problems experienced when pheromaxein subunits exist separately. PheroA/trx and pheroC/trx were therefore mixed together and cleaved from thioredoxin simultaneously so that pheromaxein subunits were given an instant opportunity to associate under oxido-shuffling conditions. The glutathione oxido-shuffling system allowed the disulphide bridges between pheromaxein A and C to rearrange until the correct native structure was formed. This novel approach combines affinity purification with a coupled fusion protein-cleavage and refolding technique.     

Modulation of sulfated glycosaminoglycan and small proteoglycan synthesis by the extracellular matrix

Cell culture in collagen lattice is known to be a more physiological model than monolayer for studying the regulation of extracellular matrix protein deposition. The synthesis of sulfated glycosaminoglycans (GAG) and dermatan sulfate (DS) proteoglycans by 3 cell strains were studied in confluent monolayers grown on plastic surface, in comparison to fully retracted collagen lattices. Cells were labelled with³⁵S-sulfate, followed by GAG and proteoglycan analysis by cellulose acetate and SDS-polyacrylamide gel electrophoresis, respectively. The 3 cell strains contracted the lattice in a similar way. In monolayer cultures, the major part of GAG was secreted into culture medium whereas in lattice cultures of dermal fibroblasts and osteosarcoma MG-63 cells but not fibrosarcoma HT-1080 cells, a higher proportion of GAGs, including dermatan sulfate, was retained within the lattices. Small DS proteoglycans, decorin and biglycan, were detected in fibroblasts and MG-63 cultures. They were preferentially trapped within the collagen gel. In retracted lattices, decorin had a higher M_r than in monolayer. Biglycan was detected in monolayer and lattice cultures of MG-63 cells but in lattice cultures only in the case of fibroblasts. In this last case, an up regulation of biglycan mRNA steady state level and down regulation of decorin mRNA was observed, in comparison to monolayers, indicating that collagen can modulate the phenotypical expression of small proteoglycan genes.Supported by a fellowship from the Centre National de la Recherche Scientifique     

Phylogeography of a Marine Insular Endemic in the Atlantic Macaronesia: The Azorean Barnacle,Megabalanus azoricus (Pilsbry, 1916)

The Azorean barnacle, Megabalanus azoricus (Pilsbry, 1916), is a Macaronesian endemic whose obscure taxonomy and the unknown relationships among forms inhabiting isolated Northern Atlantic oceanic islands is investigated by means of molecular analysis herein. Mitochondrial data from the 16S rRNA and COX1 genes support its current species status, tropical ancestry, and the taxonomic homogeneity throughout its distribution range. In contrast, at the intraspecific level and based on control region sequences, we detected an overall low level of genetic diversity and three divergent lineages. The haplogroups α and γ were sampled in the Azores, Madeira, Canary, and Cabo Verde archipelagos; whereas haplogroup β was absent from Cabo Verde. Consequently, population analysis suggested a differentiation of the Cabo Verde population with respect to the genetically homogenous northern archipelagos generated by current oceanographic barriers. Furthermore, haplogroup α, β, and γ demographic expansions occurred during the interglacial periods MIS5 (130 Kya - thousands years ago -), MIS3 (60 Kya), and MIS7 (240 Kya), respectively. The evolutionary origin of these lineages is related to its survival in the stable southern refugia and its demographic expansion dynamics are associated with the glacial-interglacial cycles. This phylogeographic pattern suggests the occurrence of genetic discontinuity informative to the delimitation of an informally defined biogeographic entity, Macaronesia, and its generation by processes that delineate genetic diversity of marine taxa in this area.     

Extracellular superoxide dismutase protects cardiovascular syndecan-1 from oxidative shedding

The extracellular matrix is a complex system that regulates cell function within a tissue. The antioxidant enzyme extracellular superoxide dismutase (EC-SOD) is bound to the matrix, and previous studies show that a lack of EC-SOD results in increased cardiac injury, fibrosis, and loss of cardiac function. This study tests the hypothesis that EC-SOD protects against cardiac fibrosis mechanistically by limiting oxidative stress and oxidant-induced shedding of syndecan-1 in the extracellular matrix. Wild-type and EC-SOD null mice were treated with a single dose of doxorubicin, 15 mg/kg, and evaluated on day 15. Serum and left-ventricle tissue were collected for biochemical assays, including Western blot, mRNA expression, and immunohistochemical staining for syndecan-1. The loss of EC-SOD and doxorubicin-induced oxidative injury led to increases in shed syndecan-1 in the serum, which originates from the endothelium of the vasculature. The shed syndecan-1 ectodomain induces proliferation of primary mouse cardiac fibroblasts. This study suggests that one mechanism by which EC-SOD protects the heart against cardiac fibrosis is the prevention of oxidative shedding of cardiovascular syndecan-1 and its subsequent induction of fibroblast proliferation. This study provides potential new targets for understanding and altering fibrosis progression in the heart.