Binding of endostatin to phosphatidylserine-containing membranes and formation of amyloid-like fibers

Endostatin, the 20-kDa C-terminal NC1 domain of collagen XVIII, is an endogenous inhibitor of tumor angiogenesis and tumor growth. A major problem in reconciling the many reported in vitro effects of endostatin is the lack of a high-affinity receptor, and a search for the latter continues. In accordance with the above, the molecular mechanisms of action of endostatin remain elusive. We show here that endostatin binds to membranes containing acidic phospholipids, phosphatidylserine (PS) or phosphatidylglycerol (PG). More specifically, a red shift in the fluorescence emission of Trp of endostatin in the presence of liposomes containing these anionic lipids was evident, revealing the average environment of Trps to become less hydrophobic. This shift was not observed for phosphatidylcholine (PC) liposomes, demonstrating the acidic lipid to be required. Quenching by endostatin of the fluorescence of a pyrene-labeled phospholipid analogue in PS containing membranes was seen, while there was no effect for PC liposomes. Resonance energy transfer from the Trp residues of endostatin to a dansyl-labeled phospholipid further confirmed the association of endostatin with PS-containing membranes, whereas there was no binding to PC liposomes. Intriguingly, the association of endostatin with PS-containing liposomes triggered the formation of fibers, with Congo red staining producing green birefringence characteristic for amyloid. Lipid was incorporated into these fibers, as shown by staining when a trace amount (X = 0.02) of fluorescent phospholipid analogues was present in the liposomes. No fiber formation was seen when endostatin was added to liposomes composed of PC only. Because PS has been reported to be exposed in the outer surface of the plasma membrane of cancer cells and vascular endothelial cells, our results suggest that this lipid could represent a target for endostatin in the cancer cell surface and tumors, thus suggesting a novel mechanism of its action. More specifically, analogous to a number of other cytotoxic proteins interacting with negatively charged lipids, PS-triggered fiber formation by endostatin on the surface of cancer cells would impair the permeability barrier function of the plasma membrane, resulting in cell death.     

Activation of innate immunity, inflammation, and potentiation of DNA vaccination through mammalian expression of the TLR5 agonist flagellin

Improving DNA vaccination remains a fundamental goal in vaccine research. Theoretically, this could be achieved by molecules encoded by DNA capable of activating TLRs to mimic inflammatory responses generated by infection. Therefore, we constructed an expression vector that allows mammalian cells to express the TLR5 agonist flagellin (FliC) at the cell surface. In vitro, cell lines expressing FliC stimulated production of proinflammatory cytokines and the up-regulation of costimulatory molecules on monocytes. Mice given the FliC expression vector intradermally exhibited site-specific inflammation and, in combination with vectors expressing Ags, developed dramatic increases in Ag-specific IgG as well as IgA. Surprisingly, mice also developed strong Ag-specific MHC class I-restricted cellular immunity. To determine whether vaccination using FliC vectors could elicit protective immunity to an infectious agent, mice were given dermal injections of FliC expression vector together with a vector encoding the influenza A virus nucleoprotein. This vaccination strategy elicited protective immunity to lethal influenza A virus infection. These results demonstrate that expression of DNA-encoded TLR agonists by mammalian cells greatly enhance and broaden immune responses, imposing new possibilities on DNA vaccination to infectious agents and cancer.     

Sequential deposition of latent TGF-beta binding proteins (LTBPs) during formation of the extracellular matrix in human lung fibroblasts

Latent TGF-beta binding proteins (LTBPs) mediate the targeting of latent TGF-beta complexes into ECM structures, which is important for TGF-beta activation and functions. LTBPs-1, -3 and -4 associate with and regulate the bioavailability of TGF-betas. We investigated whether LTBP-3 and -4 are associated with pericellular fibrillar structures of human lung fibroblast ECM, and which of their domains are important for this function. Immunoblotting analyses of isolated insoluble matrices as well as immunofluorescence analyses and confocal microscopy indicated that both LTBP-3 and -4 get assembled into the ECM. Interestingly, LTBP-4 was not detected until 7-10 days of culture and LTBP-3 until 14 days of culture. This was a major difference from the deposition kinetics of LTBP-1, which was detected already within 2 days of culture. Expression analyses by real time RT-PCR indicated that the slow appearance of LTBP-3 and -4 was due to the low expression levels soon after subculture. Recombinant N-terminal fragments of LTBP-3 and -4 bound readily to fibroblast ECM. The C-terminal domain of LTBP-4, but not of LTBP-3, also associated with the matrix structures. The levels of ECM-associated latent complexes of TGF-beta1 increased in parallel with the increased production and deposition of the LTBPs. The amount of active TGF-beta in the conditioned medium decreased during extended culture. Our results suggest that ECM is an important site of deposition also for LTBP-3 and -4 and that the temporal and spatial targeting of the TGF-beta complexes are associated with ECM maturation.     

TRIM37 defective in mulibrey nanism is a novel RING finger ubiquitin E3 ligase

Mulibrey nanism is an autosomal recessive prenatal-onset growth disorder characterized by dysmorphic features, cardiomyopathy, and hepatomegaly. Mutations in TRIM37 encoding a tripartite motif (TRIM, RING-B-box-coiled-coil)-family protein underlie mulibrey nanism. We investigated the ubiquitin ligase activity predicted for the RING domain of TRIM37 by analyzing its autoubiquitination. Full-length TRIM37 and its TRIM domain were highly polyubiquitinated when co-expressed with ubiquitin. Polyubiquitination was decreased in a mutant protein with disrupted RING domain (Cys35Ser;Cys36Ser) and in the Leu76Pro mutant protein, a disease-associated missense mutation affecting the TRIM domain of TRIM37. Bacterially produced GST-TRIM domain fusion protein, but not its Cys35Ser;Cys36Ser or Leu76Pro mutants, were polyubiquitinated in cell-free conditions, implying RING-dependent modification. Ubiquitin was also identified as an interaction partner for TRIM37 in a yeast two-hybrid screen. Ectopically expressed TRIM37 rapidly formed aggregates that were ubiquitin-, proteasome subunit-, and chaperone-positive in immunofluorescence analysis, defining them as aggresomes. The Cys35Ser;Cys36Ser mutant and the Leu76Pro and Gly322Val patient mutant proteins were markedly less prone to aggregation, implying that aggresomal targeting reflects a physiological function of TRIM37. These findings suggest that TRIM37 acts as a TRIM domain-dependent E3 ubiquitin ligase and imply defective ubiquitin-dependent degradation of an as-yet-unidentified target protein in the pathogenesis of mulibrey nanism.     

A Bayesian analysis of multivariate doubly-interval-censored dental data

A Bayesian survival analysis is presented to examine the effect of fluoride-intake on the time to caries development of the permanent first molars in children between 7 and 12 years of age using a longitudinal study conducted in Flanders. Three problems needed to be addressed. Firstly, since the emergence time of a tooth and the time it experiences caries were recorded yearly, the time to caries is doubly interval censored. Secondly, due to the setup of the study, many emergence times were left-censored. Thirdly, events on teeth of the same child are dependent. Our Bayesian analysis is a modified version of the intensity model of Harkanen et al. (2000, Scandinavian Journal of Statistics 27, 577-588). To tackle the problem of the large number of left-censored observations a similar Finnish data set was introduced. Our analysis shows no convincing effect of fluoride-intake on caries development.     

Formation of biofilms in drinking water distribution networks,a case study in two cities in Finland and Latvia

The formation of biofilms in drinking water distribution networks is a significant technical, aesthetic and hygienic problem. In this study, the effects of assimilable organic carbon, microbially available phosphorus (MAP), residual chlorine, temperature and corrosion products on the formation of biofilms were studied in two full-scale water supply systems in Finland and Latvia. Biofilm collectors consisting of polyvinyl chloride pipes were installed in several waterworks and distribution networks, which were supplied with chemically precipitated surface waters and groundwater from different sources. During a 1-year study, the biofilm density was measured by heterotrophic plate counts on R2A-agar, acridine orange direct counting and ATP-analyses. A moderate level of residual chorine decreased biofilm density, whereas an increase of MAP in water and accumulated cast iron corrosion products significantly increased biofilm density. This work confirms, in a full-scale distribution system in Finland and Latvia, our earlier in vitro finding that biofilm formation is affected by the availability of phosphorus in drinking water.     

Expression and characterization of minican, a recombinant syndecan-1 with extensively truncated core protein.

Syndecan-1 is an integral membrane heparan sulfate/chondroitin sulfate proteoglycan, involved in the control of cell growth and differentiation. The biological activities of syndecan-1 involve interactions with a variety of extracellular ligands, such as growth factors and matrix components, that are mainly mediated by the heparan sulfate moieties. The expression of syndecan-1 is downregulated in various malignant tumors, and low levels of expression appear to correlate with poor prognosis of some cancer types. On the other hand, the extracellular portion of syndecan-1 (ectodomain) has been demonstrated to inhibit the proliferation of various cancer cells in culture, suggesting that proteoglycan-like molecules should be studied further with regard to their antitumor activities. We have expressed, in CHO cells, a truncated syndecan-1 ectodomain ("minican") harboring domains for glycosaminoglycan attachment and antibody recognition. Analysis of recombinant minican indicates that it shares some of the biochemical and biological characteristics attributed to syndecan-1 ectodomain. Minican was thus substituted with heparan sulfate chains and bound to extracellular matrix proteins as well as fibroblast growth factors. Notably, minican inhibited the proliferation of S115 mouse mammary carcinoma cells and the effect seemed to involve inhibition of the Ras/Erk signaling pathway. Our data suggest that recombinant syndecan-1 with a minimal protein component is biologically active. This information may provide useful in further design of proteoglycan-like antitumor molecules.     

Landscape context affects the success of habitat restoration: large‐scale colonization patterns of saproxylic and fire‐associated species in boreal forests

Aim Restoration of habitats may be used as a conservation tool when ecosystems have lost their natural structure, dynamics or functioning over large areas. Controlled and planned use of fire could be an effective way to restore habitats of many threatened species in boreal forests where fire suppression has been effective. We asked whether the large‐scale landscape context affects the occurrence of rare and threatened species in forest habitats that have been burned to restore their fire‐related structures. Location Boreal forests in southern Finland. Methods We designed a large‐scale field experiment that included nine Pinus sylvestris forests (5–10 ha each) in southern Finland. Sites were located in two regions: (1) in eastern region with shorter management history and (2) in western region where intensive forestry has continued longer. We evaluated whether restoration of dead/burned wood is beneficial for rare and conservation‐dependent species and measured the recovery of pyrophilous and red‐listed insects (beetles and flatbugs) in burned forests, using standardized sampling effort. Altogether, 956 individuals of 29 red‐listed and pyrophilous species were sampled. Results Rare species colonized areas quickly, but there was a clear difference in species richness between the regions. The eastern forests harboured higher species richness after restoration. In these sites, the average species richness was 13.7 species per site, whereas in western forests it was 5.0 species per site. Similar pattern was also observed in subgroups: the corresponding numbers for pyrophilous species were 9.7 vs. 3.8, for red‐listed 8.7 vs. 2.3 and for red‐listed pyrophiles 4.7 vs. 1.2. Main conclusions Introducing fire back to boreal forests can aid in the recovery of rare species, but the landscape context considerably affects the success of restoring species. If restored habitats are located in landscapes that have lost their natural properties long ago, the success of restoration seems to be more challenging than in landscapes where habitats have been modified more recently.     

Occurrence of cysteine proteinase inhibitors in cells of the monocytic-histiocytic system. An immunohistochemical investigation

Human tissues are known to contain two low molecular weight (MW about 12,000) cysteine proteinase inhibitors, i.e. an acid inhibitor (ACPI) with pI 4.7-5.0 and a neutral inhibitor (NCPI) with pI 6.0-6.5. ACPI is abundant in cornifying epithelial tissues and in the dendritic reticulum cells of germinal centres of the lymph nodes. NCPI is abundant in lymphatic tissue and is known to be synthesized and released by mononuclear phagocytes. In this report NCPI was localized immunohistochemically in the epitheloid cells of most sarcoidotic lymph nodes, in lymph node macrophages after lymphangiography and in alveolar macrophages, while no ACPI could be demonstrated in the same cells by similar methods. These inhibitors were not demonstrable in lymph node sinus histiocytosis. Peripheral blood monocytes did not exhibit any NCPI immunoreactivity. In occasional blood monocytes anti-ACPI serum gave a weak reaction, the specificity of which is questionable. These data suggest that studies on cysteine proteinase inhibitors reveal basic differences in the various histiomonocytic cells and possibly differences in their functional stages.