Specific induction of secreted proteins by transforming growth factor-beta and 12-O-tetradecanoylphorbol-13-acetate. Relationship with an inhibitor of plasminogen activator

To examine the mechanisms by which transforming growth factors (TGFs) regulate the proliferation of eukaryotic cells, five cell lines, from different species and tissues, were treated with three agents that inhibit DNA synthesis and proliferation: BSC-1 cell-derived growth inhibitor (GI/TGF-beta), platelet-derived transforming growth factor-beta (TGF-beta), and 12-O-tetradecanoylphorbol-13-acetate. The cell lines tested were mink lung CCL 64 epithelial cells, Maloney sarcoma virus-transformed CCL 64.1, monkey kidney BSC-1 epithelial cells, human epidermoid A431 cells, and mouse embryo AKR-2B (clone 84A) cells. All cell lines responded to one or more of these agents by synthesizing and secreting a 48 to 51-kDa protein (IIP48). The TGF-beta s and 12-O-tetradecanoylphorbol-13-acetate had little or no effect on the incorporation of [35S] methionine into other secreted proteins or on the pattern of [35S]methionine-labeled intracellular proteins analyzed by one-dimensional, sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The maximum increase in induction of IIP48 varied from 2-fold to greater than 800-fold compared with the controls and occurred within 6 h of adding GI/TGF-beta to CCL 64 cells. Actinomycin D, alpha-amanitin, or 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole selectively decreased both the control and induced levels of IIP48 even after as little as 6 h of incubation. Thus, it appears that IIP48 mRNA turns over rapidly. Induction of IIP48 was dissociated from the inhibition of DNA synthesis by GI/TGF-beta. However, we found that epidermal growth factor and GI/TGF-beta act synergistically to increase the secreted level of IIP48. Others have shown that epidermal growth factor and TGF-beta act synergistically to stimulate growth of cells in agar. IIP48 from CCL 64, BSC-1, and AKR-2B cells is specifically immunoprecipitated by antibody to bovine plasminogen activator inhibitor. We found previously that TGF-beta also inhibits the production of major excreted protein, a thiol protease. It is proposed that TGF-beta is able to promote anchorage-independent growth of untransformed cells because of its ability to inhibit the production of secreted proteases and to increase the production of protease inhibitors.     

Uptake of alpha-aminoisobutyric acid and phosphate by membrane vesicles derived from growing and quiescent fibroblasts.

Membrane vesicles derived principally from the plasma membrane and endoplasmic reticulum of mouse 3T3 cells transformed by Simian virus 40 take up alpha-aminoisobutyric acid (AIB) and phosphate (Pi). When NaCl is added simultaneously with AIB or Pi, uptake rises two- to three-times above the equilibrium to accumulate AIB or Pi over the control value, in the presence of a Na+ gradient, is almost lost in membrane vesicles derived from benzpyrene-transformed 3T3 cells (BP3T3) arrested in the G1 phase of the cell cycle by serum starvation. When added to the membranes with NaCl and the uptake substrate, a combination of fibroblast growth factor (FGF) and epidermal growth factor EGF restores the ability of the membranes to accumulate AIB and Pi over the control value.     

Specific Chaperones for the Type VII Protein Secretion Pathway

Mycobacteria use the dedicated type VII protein secretion systems ESX-1 and ESX-5 to secrete virulence factors across their highly hydrophobic cell envelope. The substrates of these systems include the large mycobacterial PE and PPE protein families, which are named after their characteristic Pro-Glu and Pro-Pro-Glu motifs. Pathogenic mycobacteria secrete large numbers of PE/PPE proteins via the major export pathway, ESX-5. In addition, a few PE/PPE proteins have been shown to be exported by ESX-1. It is not known how ESX-1 and ESX-5 recognize their cognate PE/PPE substrates. In this work, we investigated the function of the cytosolic protein EspG(5), which is essential for ESX-5-mediated secretion in Mycobacterium marinum, but for which the role in secretion is not known. By performing protein co-purifications, we show that EspG(5) interacts with several PPE proteins and a PE/PPE complex that is secreted by ESX-5, but not with the unrelated ESX-5 substrate EsxN or with PE/PPE proteins secreted by ESX-1. Conversely, the ESX-1 paralogue EspG(1) interacted with a PE/PPE couple secreted by ESX-1, but not with PE/PPE substrates of ESX-5. Furthermore, structural analysis of the complex formed by EspG(5) and PE/PPE indicates that these proteins interact in a 1:1:1 ratio. In conclusion, our study shows that EspG(5) and EspG(1) interact specifically with PE/PPE proteins that are secreted via their own ESX systems and suggests that EspG proteins are specific chaperones for the type VII pathway.     

Significant genetic admixture after reintroduction of peregrine falcon (<Emphasis Type="Italic">Falco peregrinus</Emphasis>) in Southern Scandinavia

The peregrine falcon (Falco peregrinus) population in southern Scandinavia was almost extinct in the 1970’s. A successful reintroduction project was launched in 1974, using captive breeding birds of northern and southern Scandinavian, Finnish and Scottish origin. We examined the genetic structure in the pre-bottleneck population using eleven microsatellite markers and compared the data with the previously genotyped captive breeding population and contemporary wild population. Museum specimens between 53 and 130 years old were analyzed. Despite an apparent loss of historical genetic diversity, the contemporary population shows a relatively high level of genetic variation. Considerable gene introgression from captive breeding stock used to repopulate the former range of southern Scandinavian peregrines may have altered the genetic composition of this population. Both the historical and contemporary northern and southern Scandinavian populations are genetically differentiated. The reintroduction project implemented in the region and the use of non-native genetic stock likely prevented the southern Scandinavian population from extinction and thus helped maintain the level of genetic diversity and prevent inbreeding depression. The population is rapidly increasing in numbers and range and shows no indication of reduced fitness or adaptive capabilities in the wake of the severe bottleneck and the reintroduction.     

Alkylation damage in DNA and RNA--repair mechanisms and medical significance

Alkylation lesions in DNA and RNA result from endogenous compounds, environmental agents and alkylating drugs. Simple methylating agents, e.g. methylnitrosourea, tobacco-specific nitrosamines and drugs like temozolomide or streptozotocin, form adducts at N- and O-atoms in DNA bases. These lesions are mainly repaired by direct base repair, base excision repair, and to some extent by nucleotide excision repair (NER). The identified carcinogenicity of O(6)-methylguanine (O(6)-meG) is largely caused by its miscoding properties. Mutations from this lesion are prevented by O(6)-alkylG-DNA alkyltransferase (MGMT or AGT) that repairs the base in one step. However, the genotoxicity and cytotoxicity of O(6)-meG is mainly due to recognition of O(6)-meG/T (or C) mispairs by the mismatch repair system (MMR) and induction of futile repair cycles, eventually resulting in cytotoxic double-strand breaks. Therefore, inactivation of the MMR system in an AGT-defective background causes resistance to the killing effects of O(6)-alkylating agents, but not to the mutagenic effect. Bifunctional alkylating agents, such as chlorambucil or carmustine (BCNU), are commonly used anti-cancer drugs. DNA lesions caused by these agents are complex and require complex repair mechanisms. Thus, primary chloroethyl adducts at O(6)-G are repaired by AGT, while the secondary highly cytotoxic interstrand cross-links (ICLs) require nucleotide excision repair factors (e.g. XPF-ERCC1) for incision and homologous recombination to complete repair. Recently, Escherichia coli protein AlkB and human homologues were shown to be oxidative demethylases that repair cytotoxic 1-methyladenine (1-meA) and 3-methylcytosine (3-meC) residues. Numerous AlkB homologues are found in viruses, bacteria and eukaryotes, including eight human homologues (hABH1-8). These have distinct locations in subcellular compartments and their functions are only starting to become understood. Surprisingly, AlkB and hABH3 also repair RNA. An evaluation of the biological effects of environmental mutagens, as well as understanding the mechanism of action and resistance to alkylating drugs require a detailed understanding of DNA repair processes.     

Lipophilic Prodrugs and Formulations of Conventional (Deoxy)Nucleoside and Fluoropyrimidine Analogs in Cancer

Many drugs that are currently used for the treatment of cancer have limitations, such as induction of resistance and/or poor biological half-life, which reduce their clinical efficacy. To overcome these limitations, several strategies have been explored. Chemical modification by the attachment of lipophilic moieties to (deoxy)nucleoside analogs should enhance the plasma half-life, change the biodistribution, and improve cellular uptake of the drug. Attachment of a lipophilic moiety to a phosphorylated (deoxy)nucleoside analog will improve the activity of the drugs by circumventing the rate-limiting activation step of (deoxy)nucleoside analogs. Encapsulating drugs in nanoparticles or liposomes protects the drug against enzymatic breakdown in the plasma and makes it possible to get lipophilic compounds to the tumor site. In this review, we discuss the considerable progress that has been made in increasing the efficacy of classic (deoxy)nucleoside and fluoropyrimidine compounds by chemical modifications and alternative delivery systems.     

Indirect effects of domestic and wild herbivores on butterflies in an African savanna

Indirect interactions driven by livestock and wild herbivores are increasingly recognized as important aspects of community dynamics in savannas and rangelands. Large ungulate herbivores can both directly and indirectly impact the reproductive structures of plants, which in turn can affect the pollinators of those plants. We examined how wild herbivores and cattle each indirectly affect the abundance of a common pollinator butterfly taxon, Colotis spp., at a set of long‐term, large herbivore exclosure plots in a semiarid savanna in central Kenya. We also examined effects of herbivore exclusion on the main food plant of Colotis spp., which was also the most common flowering species in our plots: the shrub Cadaba farinosa. The study was conducted in four types of experimental plots: cattle‐only, wildlife‐only, cattle and wildlife (all large herbivores), and no large herbivores. Across all plots, Colotis spp. abundances were positively correlated with both Cadaba flower numbers (adult food resources) and total Cadaba canopy area (larval food resources). Structural equation modeling (SEM) revealed that floral resources drove the abundance of Colotis butterflies. Excluding browsing wildlife increased the abundances of both Cadaba flowers and Colotis butterflies. However, flower numbers and Colotis spp. abundances were greater in plots with cattle herbivory than in plots that excluded all large herbivores. Our results suggest that wild browsing herbivores can suppress pollinator species whereas well‐managed cattle use may benefit important pollinators and the plants that depend on them. This study documents a novel set of ecological interactions that demonstrate how both conservation and livelihood goals can be met in a working landscape with abundant wildlife and livestock.     

New Production Regulates Export Stoichiometry in the Ocean

The proportion in which carbon and growth-limiting nutrients are exported from the oceans’ productive surface layer to the deep sea is a crucial parameter in models of the biological carbon pump. Based on >400 vertical flux observations of particulate organic carbon (POC) and nitrogen (PON) from the European Arctic Ocean we show the common assumption of constant C:N stoichiometry not to be met. Exported POC:PON ratios exceeded the classical Redfield atomic ratio of 6.625 in the entire region, with the largest deviation in the deep Central Arctic Ocean. In this part the mean exported POC:PON ratio of 9.7 (a:a) implies c. 40% higher carbon export compared to Redfield-based estimates. When spatially integrated, the potential POC export in the European Arctic was 10–30% higher than suggested by calculations based on constant POC:PON ratios. We further demonstrate that the exported POC:PON ratio varies regionally in relation to nitrate-based new production over geographical scales that range from the Arctic to the subtropics, being highest in the least productive oligotrophic Central Arctic Ocean and subtropical gyres. Accounting for variations in export stoichiometry among systems of different productivity will improve the ability of models to resolve regional patterns in carbon export and, hence, the oceans’ contribution to the global carbon cycle will be predicted more accurately.