Molecular Interplay between Endostatin, Integrins, and Heparan Sulfate

Endostatin is an endogenous inhibitor of angiogenesis. Although several endothelial cell surface molecules have been reported to interact with endostatin, its molecular mechanism of action is not fully elucidated. We used surface plasmon resonance assays to characterize interactions between endostatin, integrins, and heparin/heparan sulfate. α5β1 and αvβ3 integrins form stable complexes with immobilized endostatin (K_D = ∼1.8 × 10⁻⁸ m, two-state model). Two arginine residues (Arg²⁷ and Arg¹³⁹) are crucial for the binding of endostatin to integrins and to heparin/heparan sulfate, suggesting that endostatin would not bind simultaneously to integrins and to heparan sulfate. Experimental data and molecular modeling support endostatin binding to the headpiece of the αvβ3 integrin at the interface between the β-propeller domain of the αv subunit and the βA domain of the β3 subunit. In addition, we report that α5β1 and αvβ3 integrins bind to heparin/heparan sulfate. The ectodomain of the α5β1 integrin binds to haparin with high affinity (K_D = 15.5 nm). The direct binding between integrins and heparin/heparan sulfate might explain why both heparan sulfate and α5β1 integrin are required for the localization of endostatin in endothelial cell lipid rafts.Endostatin is an endogenous inhibitor of angiogenesis that inhibits proliferation and migration of endothelial cells (1–3). This C-fragment of collagen XVIII has also been shown to inhibit 65 different tumor types and appears to down-regulate pathological angiogenesis without side effects (2). Endostatin regulates angiogenesis by complex mechanisms. It modulates embryonic vascular development by enhancing proliferation, migration, and apoptosis (4). It also has a biphasic effect on the inhibition of endothelial cell migration in vitro, and endostatin therapy reveals a U-shaped curve for antitumor activity (5, 6). Short term exposure of endothelial cells to endostatin may be proangiogenic, unlike long term exposure, which is anti-angiogenic (7). The effect of endostatin depends on its concentration and on the type of endothelial cells (8). It exerts the opposite effects on human umbilical vein endothelial cells and on endothelial cells derived from differentiated embryonic stem cells. Furthermore, two different mechanisms (heparin-dependent and heparin-independent) may exist for the anti-proliferative activity of endostatin depending on the growth factor used to induce cell proliferation (fibroblast growth factor 2 or vascular endothelial growth factor). Its anti-proliferative effect on endothelial cells stimulated by fibroblast growth factor 2 is mediated by the binding of endostatin to heparan sulfate (9), whereas endostatin inhibits vascular endothelial growth factor-induced angiogenesis independently of its ability to bind heparin and heparan sulfate (9, 10). The broad range of molecular targets of endostatin suggests that multiple signaling systems are involved in mediating its anti-angiogenic action (11), and although several endothelial cell surface molecules have been reported to interact with endostatin, its molecular mechanisms of action are not as fully elucidated as they are for other endogenous angiogenesis inhibitors (11).Endostatin binds with relatively low affinity to several membrane proteins including α5β1 and αvβ3 integrins (12), heparan sulfate proteoglycans (glypican-1 and -4) (13), and KDR/Flk1/vascular endothelial growth factor receptor 2 (14), but no high affinity receptor(s) has been identified so far. The identification of molecular interactions established by endostatin at the cell surface is a first step toward the understanding of the mechanisms by which endostatin regulates angiogenesis. We have previously characterized the binding of endostatin to heparan sulfate chains (9). In the present study we have focused on characterizing the interactions between endostatin, α5β1, αvβ3, and αvβ5 integrins and heparan sulfate. Although interactions between several integrins and endostatin have been studied previously in solid phase assays (12) and in cell models (12, 15, 16), no molecular data are available on the binding site of endostatin to the integrins. We found that two arginine residues of endostatin (Arg²⁷ and Arg¹³⁹) participate in binding to integrins and to heparan sulfate, suggesting that endostatin is not able to bind simultaneously to these molecules displayed at the cell surface. Furthermore, we have demonstrated that α5β1, αvβ3, and αvβ5 integrins bind to heparan sulfate. This may explain why both heparan sulfate and α5β1 integrins are required for the localization of endostatin in lipid rafts, in support of the model proposed by Wickström et al. (15).     

Effect of diet and mating status on ovarian development and oviposition in the polyphagous predator Macrolophus caliginosus (Heteroptera: Miridae)

The mirid bug Macrolophus caliginosus is commercially reared on eggs of Ephestia kuehniella, constituting an effective but expensive factitious food. Artificial diets can decrease the rearing costs of this natural enemy, but developing and evaluating an artificial diet is a very time-consuming activity. In the current study, development and reproduction of M. caliginosus on two artificial diets based on egg yolk were investigated. The artificial diets resulted in longer development and lower adult weights, but survival was comparable with that of control insects fed E. kuehniella eggs. Reproductive potential of the predator reared on factitious and artificial foods was assessed using a dissection method. The influence of nymphal food on fecundity was less important than that of adult food. Adults fed E. kuehniella eggs had a preoviposition period of about 4 days, whereas adults offered only plant material started laying eggs about 7 days after emergence. Ovarian scores at day 7 were higher for females fed E. kuehniella eggs than for those given access only to a tobacco leaf. Ovarian scores were not significantly affected by mating status. In a final test, a parallel comparison of two methods for assessing reproductive response to diet was made. Here, adult couples were offered one of four diets: E. kuehniella eggs, one of two artificial diets or no food. Half of the females were dissected and the other half was held for determining lifetime oviposition. Females fed E. kuehniella eggs had superior ovarian scores and laid more eggs than those fed either artificial diet or those given no extra food. A good correlation (r = 0.97) was obtained between ovarian scores and oviposition data, indicating that dissecting females after 1 week provides a reliable estimate of fecundity as affected by diet quality. Rapid reproductive assessments as used in the current study will help to increase the rate of development of artificial diets and may contribute to more cost effective production methods for augmentative biological control agents.     

Migration speeds and orientation of Atlantic salmon and sea trout post-smolts in a Norwegian fjord system

We recorded the observed and actual swimming speeds of Atlantic salmon and sea trout post-smolts in a Norwegian fjord system, and initiated studies on the orientation mechanisms of the post-smolts. We tracked Atlantic salmon and sea trout with acoustic transmitters for up to 14 h after release. The actual swimming speed and direction of a fish relative to the ground is the vector sum of the observed movements of the fish and the movements of the water. We determined actual swimming speeds and directions of the post-smolts, which reflect their real swimming capacities and orientation, by corrections for the speed and direction of the water current. The post-smolts were actively swimming. The observed direction of movement was dependent on the actual movement of the fish and not the water current. Water currents were not systematically used as an orientation cue either in Atlantic salmon or sea trout, as the actual movements were random compared to the direction of the water current. The actual movement of sea trout were in all compass directions, with no systematic pattern. The Atlantic salmon also moved in all compass directions, but with the lowest frequency of actual movement towards the fjord.     

Functional Anatomy of the Ovule in Broad Bean (Vicia faba L.): Ultrastructural Seed Development and Nutrient Pathways

Ovules of broad bean (Vicia faba L.) were studied to discloseultrastructural features, which can facilitate nutrient transportto the embryo sac from 10 d after pollination (DAP) to the matureseed. Fertilization occurs during the first 24 h after pollination.The endosperm is a coenocyte, which is eventually consumed bythe embryo. By 10 DAP the inner integument is degraded and theouter integument adjoins the embryo sac boundary. The heart-shapedembryo approaches the embryo sac boundary at two sites, whichhere are named contact zones. Small integument cells in theneighbourhood of the first formed contact zones become separatedby prominent intercellular spaces. A heterogenous scatteringmaterial, probably representing secretion products accumulatesin these spaces. By 14-16 DAP the integument exudate disappears,and the suspensor degenerates. As the contact zones increasein size, wall ingrowths form a bridging network in the narrowspace between the embryo sac boundary and the extra-embryonicpart of the endosperm wall. The epidermal cells of the embryoseparate adjacent to these zones, and develop conspicuous wallingrowths. At 20 DAP vacuoles showing various stages in formationof protein bodies appear in the cells of the embryo.Copyright1994, 1999 Academic Press Vicia faba, broad beans, ovule, seed, nutrient transport     

A conserved ClpP‐like protease involved in spore outgrowth in Bacillus subtilis

Germination and outgrowth of endospores of the Gram‐positive bacterium Bacillus subtilis involves the degradation and conversion to free amino acids of abundant proteins located in the spore core known as small acid‐soluble proteins (SASP). This degradation is mediated primarily by the germination protease Gpr. Here we show that YmfB, a distant homologue of ClpP serine proteases that is highly conserved among endospore‐forming bacteria, contributes to SASP degradation but that its function is normally masked by Gpr. Spores from a ymfB gpr double mutant were more delayed in spore outgrowth and more impaired in SASP degradation than were spores from a gpr single mutant. The activity of YmfB relied on three putative active‐site residues as well as on the product of a small gene ylzJ located immediately downstream of, and overlapping with, ymfB. We propose that YmfB is an orphan ClpP protease that is dedicated to the degradation of a specialized family of small protein substrates.     

Angiopoietins assemble distinct Tie2 signalling complexes in endothelial cell-cell and cell-matrix contacts

The receptor tyrosine kinase Tie2, and its activating ligand Angiopoietin-1 (Ang1), are required for vascular remodelling and vessel integrity, whereas Ang2 may counteract these functions. However, it is not known how Tie2 transduces these different signals. Here, we show that Ang1 induces unique Tie2 complexes in mobile and confluent endothelial cells. Matrix-bound Ang1 induced cell adhesion, motility and Tie2 activation in cell-matrix contacts that became translocated to the trailing edge in migrating endothelial cells. In contrast, in contacting cells Ang1 induced Tie2 translocation to cell-cell contacts and the formation of homotypic Tie2-Tie2 trans-associated complexes that included the vascular endothelial phosphotyrosine phosphatase, leading to inhibition of paracellular permeability. Distinct signalling proteins were preferentially activated by Tie2 in the cell-matrix and cell-cell contacts, where Ang2 inhibited Ang1-induced Tie2 activation. This novel type of cellular microenvironment-dependent receptor tyrosine kinase activation may explain some of the effects of angiopoietins in angiogenesis and vessel stabilization.     

Species specificity of iron delivery in hybridomas

Summary Studies with Human x Human (HxH), Human x Mouse (HxM), and Mouse x Mouse (MxM) hybridomas have enabled us to define specific factors that affect hybridoma growth in a species-specific manner. Three transferrins and three lipophilic iron chelates have been tested for their ability to support hybridoma proliferation and antibody production. The results of these studies demonstrate that HxH hybridomas do not respond to bovine transferrin a⁺ concentrations up to 100 μg/ml and are approximately 100-fold less responsive to mouse transferrin than to human transferrin. HxM and MxM hybridomas respond equally to human or mouse transferrin but are 100-fold less sensitive to bovine transferrin. An antibody to the human transferrin receptor inhibited the growth-promoting activity of human or mouse transferrin on HxH hybridomas but was ineffective on HxM hybridomas. This semonstrated the functionality of the human transferrin receptor in HxH hybridomas and that human, mouse, and bovine transferrin were interacting through the mouse transferrin receptor in HxM hybridomas. HxH and HxM hybridomas respond similarly to three different iron chelates exhibiting 80 to 110% of the growth response to human transferrin. MxM hybridomas fail to respond to the iron chelates at similar concentrations, suggesting that the human genome present in the other hybridoma species confers a unique ability for utilizing iron when delivered in this form.     

Two-Dimensional Nanoscale Structural and Functional Imaging in Individual Collagen Type I Fibrils

The piezoelectric properties of single collagen type I fibrils in fascia were imaged with sub-20 nm spatial resolution using piezoresponse force microscopy. A detailed analysis of the piezoresponse force microscopy signal in controlled tip-fibril geometry revealed shear piezoelectricity parallel to the fibril axis. The direction of the displacement is preserved along the whole fiber length and is independent of the fiber conformation. It is shown that individual fibrils within bundles in skeletal muscle fascia can have opposite polar orientations and are organized into domains, i.e., groups of several fibers having the same polar orientation. We were also able to detect piezoelectric activity of collagen fibrils in the high-frequency range up to 200 kHz, suggesting that the mechanical response time of biomolecules to electrical stimuli can be ∼5 μs.     

Breeding and selection of Buxus for resistance to Calonectria pseudonaviculata

Box blight is a widespread disease of Buxus caused by the pathogen Calonectria pseudonaviculata. It is responsible for significant losses in nurseries, gardens and wild boxwood populations. Our goal was to maximize the efficiency of a breeding programme towards increased disease resistance. The use of artificial inoculation of young F1 seedlings with C. pseudonaviculata spores under greenhouse conditions appeared to be a reliable tool for early selection of interesting prebreeding material. Overall, the four hybrid populations screened showed a segregating behaviour between their parents when determining the percentage of diseased leaves and lesion diameter. Genotypes were also found with an increased tolerance as compared to the parental species. Approximately 50% of the seedlings had the same score for both parameters after artificial inoculation in the greenhouse and in the field. Of the seedlings that showed severe symptoms in the greenhouse, <15% showed no disease symptoms in the field. Therefore, for larger breeding programmes, we propose a two‐step selection procedure: first artificial inoculation at seedling level to eliminate all genotypes with severe symptoms and then evaluation of the remaining seedlings in the field. Using this strategy, we were able to select several genotypes in our four hybrid populations with improved resistance to C. pseudonaviculata.