LATE MERISTEM IDENTITY2 acts together with LEAFY to activate APETALA1.

The switch from producing vegetative structures (branches and leaves) to producing reproductive structures (flowers) is a crucial developmental transition that significantly affects the reproductive success of flowering plants. In Arabidopsis, this transition is in large part controlled by the meristem identity regulator LEAFY (LFY). The molecular mechanisms by which LFY orchestrates a precise and robust switch to flower formation is not well understood. Here, we show that the direct LFY target LATE MERISTEM IDENTITY2 (LMI2) has a role in the meristem identity transition. Like LFY, LMI2 activates AP1 directly; moreover, LMI2 and LFY interact physically. LFY, LMI2 and AP1 are connected in a feed-forward and positive feedback loop network. We propose that these intricate regulatory interactions not only direct the precision of this crucial developmental transition in rapidly changing environmental conditions, but also contribute to its robustness and irreversibility.

Stability, sterility, coagulation, and immunologic studies of salmon coagulation proteins with potential use for mammalian wound healing and cell engineering.

Fibrin sealants made by polymerization of fibrinogen activated by the protease thrombin have many applications in hemostasis and wound healing. In treatments of acute injury or surgical wounds, concentrated fibrin preparations mimic the initial matrix that normally prevents bleeding and acts as a scaffold for cells that initiate tissue repair. However risks of infectious disease, immunogenic reaction, and the high cost of purified human or other mammalian blood proteins limit widespread use of these materials. Purified coagulation proteins from Atlantic salmon represent a potentially safer, equally effective, and less costly alternative in part because of the low ambient temperature of these farmed animals and the absence of endogenous agents known to be infectious in mammalian hosts. This study reports rheologic measurements of lyophilized salmon fibrinogen and thrombin that demonstrate stability to prolonged storage and gamma irradiation sufficient to reduce viral loads by over five orders of magnitude. Coagulation and immunologic studies in rats and rabbits treated intraperitoneally with salmon fibrin show no deleterious effects on coagulation profiles and no cross reactivity with host fibrinogen or thrombin. The results support the potential of salmon fibrin as an alternative to mammalian proteins in clinical applications.

Bacterial endotoxin as inhibitor of the enzymatic activity of human thrombin.

Endotoxemia caused by bacterial lipopolysaccharides (LPS) deleteriously affects many aspects of hemostasis. Much of this effect is well characterized as being secondary to the LPS-mediated inflammatory response, but direct effects of LPS on coagulation factors may also contribute to disregulation of the hemostatic process. Spectrophotometric assays were used to investigate the effects of LPS from different bacteria on thrombin and plasmin activities. We found that enzymatic activity of purified thrombin, but not plasmin, decreases in the presence of endotoxin. LPS-mediated inhibition of thrombin activity can be reversed by plasma gelsolin and recombinant endotoxin-neutralizing protein. Preincubation of thrombin with LPS before platelet activation results in inhibition of aggregation and secretion. Additionally, a decrease of elastic shear moduli of fibrin gels was observed when their formation was induced with thrombin preincubated with LPS or when LPS was present in fibrinogen solutions during fibrin gel formation. When added to platelet-rich plasma, after activation with collagen, LPS-inhibited thrombin activity. LPS-mediated inhibition of thrombin activity may contribute to the hemostasis dysfunctions observed during endotoxemia.

Involvement of the Na+/H+ exchanger in membrane phosphatidylserine exposure during human platelet activation.

Platelet membrane phosphatidylserine (PS) exposure that regulates the production of thrombin represents an important link between platelet activation and the coagulation cascade. Here, we have evaluated the involvement of the Na+/H+ exchanger (NHE) in this process in human platelets. PS exposure induced in human platelets by thrombin, TRAP, collagen or TRAP+ collagen was abolished in a Na+ -free medium. Inhibition of the Na+/H+ exchanger (NHE) by 5-(N-Ethyl-N-Isopropyl) Amiloride (EIPA) reduced significantly PS exposure, whereas monensin or nigericin, which mimic or cause activation of NHE, respectively, reproduced the agonist effect. These data suggest a role for Na+ influx through NHE activation in the mechanism of PS exposure. This newly identified pathway does not discount a role for Ca2+, whose cytosolic concentration varies together with that of Na+ after agonist stimulation. Ca2+ deprivation from the incubation medium only attenuated PS exposure induced by thrombin, measured from the uptake of FM1-43 (a marker of phospholipid scrambling independent of external Ca2+). Surprisingly, removal of external Ca2+ partially reduced FM1-43 uptake induced by A23187, known as a Ca2+ ionophore. The residual effect can be attributed to an increase in [Na+]i mediated by the ionophore due to a lack of its specificity. Finally, phosphatidylinositol 4,5-bisphosphate (PIP2), previously reported as a target for Ca2+ in the induction of phospholipid scrambling, was involved in PS exposure through a regulation of NHE activity. All these results would indicate that the mechanism that results in PS exposure uses redundant pathways inextricably linked to the physio-pathological requirements of this process.

Flavonoid-mediated inhibition of actin polymerization in cold-activated platelets.

The response of human platelets to low temperature (below 15 degrees C) requires that they are stored at elevated temperatures and limits their storage time to 5 days for use in transfusion. Prolonged storage at room temperature leads to loss of platelet function and risk of septic conditions. The need for improved platelet storage is an important issue, and finding a key component allowing platelets to be maintained at low temperatures would have significant practical benefit. Developing such a component is challenging, because the process of cold-activation resembles that of a physiological agonist-mediated activation, but without a specific receptor that can be inhibited. A component preventing platelets' low temperature response will potentially inhibit their physiological function, making them less useful after transfusion. In the present study, we report that pretreatment of platelets with flavonoids before chilling prevents an increase in cytosolic calcium concentration, actin polymerization and platelet shape change. After warming, platelets that were chilled in the presence of flavonoids retain a normal shape change and aggregation response after stimulation by thrombin. Additionally, cold platelet activation does not increase platelet procoagulant activity evaluated by annexin V-FITC binding in the presence and absence of flavonoids. These data confirm the important links that exist between agonist- and cold-mediated platelet activation, suggesting a possible advantage of incorporating the use of flavonoids to allow platelet hypothermic-storage.

Inactivation of endotoxin by human plasma gelsolin.

Septic shock from bacterial endotoxin, triggered by the release of lipopolysaccharide (LPS) molecules from the outer wall of Gram-negative bacteria, is a major cause of human death for which there is no effective treatment once the complex inflammatory pathways stimulated by these small amphipathic molecules are activated. Here we report that plasma gelsolin, a highly conserved human protein, binds LPS from various bacteria with high affinity. Solid-phase binding assays, fluorescence measurements, and functional assays of actin depolymerizing effects show that gelsolin binds more tightly to LPS than it does to its other known lipid ligands, phosphatidylinositol 4,5-bisphosphate and lysophosphatidic acid. Gelsolin also competes with LPS-binding protein (LBP), a high-affinity carrier for LPS. One result of gelsolin-LPS binding is inhibition of the actin binding activity of gelsolin as well as the actin depolymerizing activity of blood serum. Simultaneously, effects of LPS on cellular functions, including cytoskeletal actin remodeling, and collagen-induced platelet activation by pathways independent of toll-like receptors (TLRs) are neutralized by gelsolin and by a peptide based on gelsolin residues 160-169 (GSN160-169) which comprise part of gelsolin's phosphoinositide binding site. Additionally, TLR-dependent NF-kappaB translocation in astrocytes appears to be blocked by gelsolin. These results show a strong effect of LPS on plasma gelsolin function and suggest that some effects of endotoxin in vivo may be mediated or inhibited by plasma gelsolin.

Nonlinear elasticity in biological gels.

The mechanical properties of soft biological tissues are essential to their physiological function and cannot easily be duplicated by synthetic materials. Unlike simple polymer gels, many biological materials--including blood vessels, mesentery tissue, lung parenchyma, cornea and blood clots--stiffen as they are strained, thereby preventing large deformations that could threaten tissue integrity. The molecular structures and design principles responsible for this nonlinear elasticity are unknown. Here we report a molecular theory that accounts for strain-stiffening in a range of molecularly distinct gels formed from cytoskeletal and extracellular proteins and that reveals universal stress-strain relations at low to intermediate strains. The input to this theory is the force-extension curve for individual semi-flexible filaments and the assumptions that biological networks composed of these filaments are homogeneous, isotropic, and that they strain uniformly. This theory shows that systems of filamentous proteins arranged in an open crosslinked mesh invariably stiffen at low strains without requiring a specific architecture or multiple elements with different intrinsic stiffness.

Antibacterial activities of rhodamine B-conjugated gelsolin-derived peptides compared to those of the antimicrobial peptides cathelicidin LL37, magainin II, and melittin.

The growing number of antibiotic-resistant bacteria necessitates the search for new antimicrobial agents and the principles by which they work. We report that cell membrane-permeant rhodamine B (RhB)-conjugated peptides based on the phosphatidylinositol-4,5-bisphosphate binding site of gelsolin can kill the gram-negative organisms Escherichia coli and Pseudomonas aeruginosa and the gram-positive organism Streptococcus pneumoniae. RhB linkage to the QRLFQVKGRR sequence in gelsolin was essential for the antibacterial function, since the unconjugated peptide had no effect on the bacteria tested. Because RhB-QRLFQVKGRR (also termed PBP10), its scrambled sequence (RhB-FRVKLKQGQR), and PBP10 synthesized from D-isomer amino acids show similar antibacterial properties, the physical and chemical properties of these derivatives appear to be more important than specific peptide folding for their antibacterial functions. The similar activities of PBP10 and all-D-amino-acid PBP10 also indicate that a specific interaction between RhB derivatives and bacterial proteins is unlikely to be involved in the bacterial killing function of PBP10. By using a phospholipid monolayer system, we found a positive correlation between the antibacterial function of PBP10, as well as some naturally occurring antibacterial peptides, and the intrinsic surface pressure activity at the hydrophobic-hydrophilic interface. Surprisingly, we observed little or no dependence of the insertion of these peptides into lipid monolayers on the phospholipid composition. These studies show that an effective antimicrobial agent can be produced from a peptide sequence with specificity to a phospholipid not found in bacteria, and comparisons with other antimicrobial agents suggest that the surface activities of these peptides are more important than specific binding to bacterial proteins or lipids for their antimicrobial functions.

Purification of salmon thrombin and its potential as an alternative to mammalian thrombins in fibrin sealants.

A method to produce highly purified thrombin from salmon blood is described, and a series of biochemical, cell biologic, and biophysical assays demonstrate the functional similarities and some differences between salmon and human thrombins. Salmon thrombin with specific activity greater than 1000 units/mg total protein can be prepared by modifications of the methods used for purification of human thrombin. Using a synthetic substrate based on the human fibrinogen A-alpha polypeptide sequence as an indicator of enzymatic activity, salmon and human thrombin preparations contain similar specific activities per mass of purified protein. Salmon thrombin activates human fibrinogen and initiates the formation of fibrin clots whose structure and rheologic properties are indistinguishable from those of human fibrin clotted by human thrombin. Salmon thrombin also activates human platelets. Approximately 10 times higher activities are needed for the same rate of platelet aggregation compared to human thrombin, and some aspects of platelet activation, most notably phosphatidylserine exposure, are diminished relative to the effects of human thrombin. This latter finding suggests that salmon thrombin may not activate all of the receptors that are targets of human thrombin, although it does appear to activate signals that are sufficient to produce normal rates of activation and aggregation as measured by conventional aggregometry. Together with the recent purification of salmon fibrinogen and its application in mammalian wound healing, the availability of salmon thrombin allows the formulation of biological sealants devoid of any exogenous mammalian proteins and so may aid the design of materials with increased safety from infectious disease transmission.