Effect of side group chemistry on the properties of biodegradable L-alanine cosubstituted polyphosphazenes

Biodegradable polyphosphazenes have been investigated for a variety of applications, such as controlled drug delivery matrixes, tissue-engineering scaffolds, membranes, and bone-type composites. In this study we have evaluated the effect of side group chemistry on the properties of biodegradable phosphazene polymers that contain ethyl alanato side groups together with ethyl glycinato, p-methylphenoxy, or p-phenylphenoxy side groups. The polymers were synthesized by a macromolecular substitution route. The molecular weights of aryloxy/amino acid ester cosubstituted polymers were much higher than the amino acid ester substituted polyphosphazenes described earlier. Polymer properties, such as glass transition temperature, hydrolytic degradation, surface wettability, tensile strength, and modulus of elasticity varied over a wide range following changes to the type of co-substituents on the polymer backbone. The glass transition temperatures varied from -10 to 35 degrees C and increased with the bulkiness of the side groups. Polymer films in phosphate buffer saline solution showed molecular weight declines ranging from 58% to >80% and mass loss ranging from 4% to 90% over a period of 7 weeks. Water contact angles for polymer films varied from 63 degrees to 107 degrees , with the highest angles for the alanine ethyl ester and p-phenylphenoxy cosubstituted polyphosphazene. The tensile strengths were in the range of 2.4-7.6 MPa and the modulus of elasticity was in the range of 31.4-455.9 MPa. Thus, in this study we have demonstrated the tunability of biodegradable polyphosphazenes to suit a range of biomedical applications.     

Isolation of two populations of sperm cells and microelectrophoresis of pairs of sperm cells from pollen tubes of tobacco (Nicotiana tabacum)

Prior research has indicated that the two sperm cells of Nicotiana tabacum are dimorphic, suggesting that they may participate in preferential fertilization during in vivo fusion with the egg and central cells. To probe the mechanism of potential preferential fertilization in this plant, it will be necessary to use modern sensitive molecular techniques. For this purpose, two individual populations of two sperm cells, constituting the Svn (associated with the vegetative nucleus) and Sua (unassociated with the vegetative nucleus), were isolated in the thousands from tobacco pollen tubes with a micromanipulator as a preliminary step toward research on gametic recognition using molecular techniques. Microelectrophoresis of paired sperm cells from a single pollen tube was conducted at different developmental stages. Sperm cells isolated from 1-, 2-, 3- and 4-cm stylar lengths migrated to the negative pole, with the Sua displaying significantly greater electrophoretic mobility than the Svn, reflecting a more positively charged cell surface on the Sua. The sperm cells isolated from 1-cm style are very sensitive to electron potential in an electrophoretic field, presumably reflecting that they are still in a young state. Differences in cell surface charge between the Sua and Svn may be related with cell fate during fertilization. Supported by National Natural Science Foundation of CHINA (30170060)     

A novel organization of ACT domains in allosteric enzymes revealed by the crystal structure of Arabidopsis aspartate kinase

Asp kinase catalyzes the first step of the Asp-derived essential amino acid pathway in plants and microorganisms. Depending on the source organism, this enzyme contains up to four regulatory ACT domains and exhibits several isoforms under the control of a great variety of allosteric effectors. We report here the dimeric structure of a Lys and S-adenosylmethionine-sensitive Asp kinase isoform from Arabidopsis thaliana in complex with its two inhibitors. This work reveals the structure of an Asp kinase and an enzyme containing two ACT domains cocrystallized with its effectors. Only one ACT domain (ACT1) is implicated in effector binding. A loop involved in the binding of Lys and S-adenosylmethionine provides an explanation for the synergistic inhibition by these effectors. The presence of S-adenosylmethionine in the regulatory domain indicates that ACT domains are also able to bind nucleotides. The organization of ACT domains in the present structure is different from that observed in Thr deaminase and in the regulatory subunit of acetohydroxyacid synthase III.     

Fibroblast growth factor 2 induced proliferation in osteoblasts and bone marrow stromal cells: a whole cell model

Fibroblast growth factor 2 (FGF2) can enhance the proliferative capacity of bone and bone marrow stromal cells; however, the mechanisms behind this effect are not well described. We present a whole-cell kinetic model relating receptor-mediated binding, internalization, and processing of FGF2 to osteoblastic proliferative response. Focusing on one of the potential signaling complex stoichiometries, we utilized experimentally measured and modeled estimated rate constants to predict in vitro proliferation and distinguish between potential binding orders. We found that piecewise assemblage of a ternary signaling complex may occur in several ways depending on the local binding environment. Using experimental data of endocytosed FGF2 as a constraint, we have also shown evidence of potential multistep processes involved in heparan-sulfate proteoglycans-bound FGF2 release, internalization, and fragment formation in conjunction with the normal metabolism of the proteoglycan.     

Reference concentrations of cholecalciferol in animals: a basis for establishing non-target exposure

Abstract

Cholecalciferol (vitamin D₃) is widely used as a vertebrate pesticide in New Zealand. However, cholecalciferol also occurs naturally in animals. Therefore, when trying to determine whether a non-target animal has been exposed to cholecalciferol baits, knowledge of the baseline cholecalciferol concentrations in the animal's plasma and tissue is required. We analysed cattle, sheep, pig, deer, dog and cat plasma and liver samples for the vitamin D₃ metabolite 25-hydroxycholecalciferol (25-OHD), a sensitive biomarker for cholecalciferol. Based on these data and a literature search we present 25-OHD reference ranges. We also examined the literature for 25-OHD concentrations in poisoned animals and compared these to the reference ranges. Where plasma and liver samples have 25-OHD concentrations at least four times higher than our reference ranges it is likely that the animal has been exposed to cholecalciferol baits. 25-OHD concentrations 10 times higher than the reference range indicate ingestion of abnormally high amounts of cholecalciferol.     

End-of-life care issues in advanced dementia

Appropriate management of advanced dementia requires it to be recognised as a terminal condition that needs palliative care. Interventions during this stage should be carefully chosen to ensure the improvement or maintenance of the quality of life of the person with dementia. Advanced care planning is an important aspect of dementia care. Carers and relatives should be educated and encouraged to actively participate in discussions related to artificial nutrition, cardiopulmonary resuscitation (CPR) and other medical interventions.     

Allosteric monofunctional aspartate kinases from Arabidopsis

Plant monofunctional aspartate kinase is unique among all aspartate kinases, showing synergistic inhibition by lysine and S-adenosyl-l-methionine (SAM). The Arabidopsis genome contains three genes for monofunctional aspartate kinases. We show that aspartate kinase 2 and aspartate kinase 3 are inhibited only by lysine, and that aspartate kinase 1 is inhibited in a synergistic manner by lysine and SAM. In the absence of SAM, aspartate kinase 1 displayed low apparent affinity for lysine compared to aspartate kinase 2 and aspartate kinase 3. In the presence of SAM, the apparent affinity of aspartate kinase 1 for lysine increased considerably, with K(0.5) values for lysine inhibition similar to those of aspartate kinase 2 and aspartate kinase 3. For all three enzymes, the inhibition resulted from an increase in the apparent K(m) values for the substrates ATP and aspartate. The mechanism of aspartate kinase 1 synergistic inhibition was characterized. Inhibition by lysine alone was fast, whereas synergistic inhibition by lysine plus SAM was very slow. SAM by itself had no effect on the enzyme activity, in accordance with equilibrium binding analyses indicating that SAM binding to aspartate kinase 1 requires prior binding of lysine. The three-dimensional structure of the aspartate kinase 1-Lys-SAM complex has been solved [Mas-Droux C, Curien G, Robert-Genthon M, Laurencin M, Ferrer JL & Dumas R (2006) Plant Cell18, 1681-1692]. Taken together, the data suggest that, upon binding to the inactive aspartate kinase 1-Lys complex, SAM promotes a slow conformational transition leading to formation of a stable aspartate kinase 1-Lys-SAM complex. The increase in aspartate kinase 1 apparent affinity for lysine in the presence of SAM thus results from the displacement of the unfavorable equilibrium between aspartate kinase 1 and aspartate kinase 1-Lys towards the inactive form.     

Miscibility of bioerodible polyphosphazene/poly(lactide-co-glycolide) blends

We have previously demonstrated the feasibility of blending bioerodible polyphosphazenes with poly(lactide-co-glycolide) (PLGA) to form versatile polymeric materials with altered bioerosion properties. These studies demonstrated the effective neutralization of the acidic degradation products of PLGA by the polyphosphazene hydrolysis products. In the present study, five new polymers of dipeptide polyphosphazenes poly[(ethyl glycinato)x(glycyl-ethyl glycinato)yphosphazene] and novel blends of these polyphosphazenes with poly(lactide-co-glycolide) (PLGA) were synthesized and fabricated. The miscibility was analyzed using differential scanning calorimetry and scanning electron microscopy. Hydrogen bonding within the blends was assessed by attenuated total reflectance infrared spectroscopy. The phosphazene component of the blend contained varying ratios of the glycyl-glycine ethyl ester to the glycine ethyl ester. Poly[(ethyl glycinato)0.5(glycine ethyl glycinato)1.5phosphazene formed completely miscible blends with PLGA (50:50) and PLGA (85:15). This is ascribed to the multiple hydrogen-bonding sites within the side groups of the polyphosphazene. The components of the blend act as plasticizers for each other because a glass transition temperature for each blend was detected at a lower temperature than for each individual polymer. A hydrolysis study showed that unblended solid poly[(ethyl glycinato)0.5(glycyl ethyl glycinato)1.5phosphazene] hydrolyzed in less than 1 week. However, the blends degraded at a slower rate than both parent polymers. This is attributed to the buffering capacity of the polyphosphazene hydrolysis products, which increases the pH of the degradation media from 2.5 to 4, thereby slowing the degradation rate of PLGA.     

Fabrication and optimization of methylphenoxy substituted polyphosphazene nanofibers for biomedical applications

Electrospinning has developed as a unique and versatile process to fabricate ultrathin fibers in the form of nonwoven meshes or as oriented arrays from a variety of polymers. The very small dimension of these fibers can generate a high surface area, which makes them potential candidates for various biomedical and industrial applications. The objective of the present study was to develop nanofibers from polyphosphazenes, a class of inorganic-organic polymers known for high biocompatibility, high-temperature stability, and low-temperature flexibility. Specifically, we evaluated the feasibility of developing bead-free nonwoven nanofiber mesh from poly[bis(p-methylphenoxy)phosphazene] (PNmPh) by electrospinning. The effect of process parameters such as nature of solvent, concentration of the polymer solution, effect of needle diameter, and applied potential on the diameter and morphology (beaded or bead-free) of resulting nanofibers were investigated. It was found that solution of PNmPh in chloroform at a concentration range of 7% (wt/v) to 9% (wt/v) can be readily electrospun to form bead-free fibers at room temperature. The mean diameter of the fibers obtained under optimized spinning condition was found to be approximately 1.2 microm. The bead-free, cylindrical nanofibers formed under the optimized condition showed a slightly irregular surface topography with indentations of a few nanometer scale. Further, the electrospun nanofiber mats supported the adhesion of bovine coronary artery endothelial cells (BCAEC) as well as promoted the adhesion and proliferation of osteoblast like MC3T3-E1 cells.