Association of candidate genes with flowering time and water-soluble carbohydrate content in Lolium perenne (L.)

Sk?t, L., Humphreys, J., Humphreys, M. O., Thorogood, D., Gallagher, J. A., Sanderson, R., Armstead, I. P., Thomas, I. D. (2007). Association of candidate genes with flowering time and water-soluble carbohydrate content in Lolium perenne (L.). Genetics, 177 (1), 535-547. Sponsorship: BBSRC RAE2008

Application of mesoporous silica for the oral delivery of poorly water-soluble drugs

The objective of this thesis was to improve the dissolution rate of the poorly waters-soluble drug, fenofibrate by processing it with a high surface area carrier, mesoporous silica. The subsequent properties of the drug – silica composite were studied in terms of drug distribution within the silica matrix, solid state and release properties. Prior to commencing any experimental work, the properties of unprocessed mesoporous silica and fenofibrate were characterised (chapter 3), this allowed for comparison with the processed samples studied in later chapters. Fenofibrate was a highly stable, crystalline drug that did not adsorb moisture, even under long term accelerated storage conditions. It maintained its crystallinity even after SC-CO2 processing. Its dissolution rate was limited and dependent on the characteristics of the particular in vitro media studied. Mesoporous silica had a large surface area and mesopore volume and readily picked up moisture when stored under long term accelerated storage conditions (75% RH, 40 oC). It maintained its mesopore character after SC-CO2 processing. A variety of methods were employed to process fenofibrate with mesoporous silica including physical mixing, melt method, solvent impregnation and novel methods such as liquid and supercritical carbon dioxide (SC-CO2) (chapter 4). It was found that it was important to break down the fenofibrate particulate structure to a molecular state to enable drug molecules enter into the silica mesopores. While all processing methods led to some increase in fenofibrate release properties; the impregnation, liquid and SC-CO2 methods produced the most rapid release rates. SC-CO2 processing was further studied with a view to optimising the processing parameters to achieve the highest drug-loading efficiency possible (chapter 5). In this thesis, it was that SC-CO2 processing pressure had a bearing on drug-loading efficiency. Neither pressure, duration or depressurisation rate affected drug solid state or release properties. The amount of drug that could be loaded onto to the mesoporous silica successfully was also investigated at different ratios of drug mass to silica surface area under constant SC-CO2 conditions; as the drug – silica ratio increased, the drug-loading efficiency decreased, while there was no effect on drug solid state or release properties. The influence of the number of drug-loading steps was investigated (chapter 6) with a view to increasing the drug-loading efficiency. This multiple step approach did not yield an increase in drug-loading efficiency compared to the single step approach. It was also an objective in this chapter to understand how much drug could be loaded into silica mesopores; a method based on the known volume of the mesopores and true density of drug was investigated. However, this approach led to serious repercussions in terms of the subsequent solid state nature of the drug and its release performance; there was significant drug crystallinity and reduced release extent. The impact of in vitro release media on fenofibrate release was also studied (chapter 6). Here it was seen that media containing HCl led to reduced drug release over time compared to equivalent media not containing HCl. The key findings of this thesis are discussed in chapter 7 and included: 1. Drug – silica processing method strongly influenced drug distribution within the silica matrix, drug solid state and release. 2. The silica surface area and mesopore volume also influenced how much drug could be loaded. It was shown that SC-CO2 processing variables such as processing pressure (13.79 – 41.37 MPa), duration time (4 – 24 h) and depressurisation rate (rapid or controlled) did not influence the drug distribution within the SBA- 15 matrix, drug solid state form or release. Possible avenues of research to be considered going forward include the development and application of high resolution imaging techniques to visualise drug molecules within the silica mesopores. Also, the issues surrounding SBA-15 usage in a pharmaceutical manufacturing environment should be addressed.

The neuropeptide pituitary adenylate cyclase activating protein stimulates human monocytes by transactivation of the Trk/NGF pathway.

Transactivation is a process whereby stimulation of G-protein-coupled receptors (GPCR) activates signaling from receptors tyrosine kinase (RTK). In neuronal cells, the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) acting through the GPCR VPAC-1 exerts trophic effects by transactivating the RTK TrkA receptor for the nerve growth factor (NGF). Both PACAP and NGF have pro-inflammatory activities on monocytes. We have tested the possibility that in monocytes, PACAP, as reported in neuronal cells, uses NGF/TrkA signaling pathway. In these cells, PACAP increases TrkA tyrosine phosphorylations through a PI-3kinase dependent but phospholipase C independent pathway. K252a, an inhibitor of TrkA decreases PACAP-induced Akt and ERK phosphorylation and calcium mobilisation resulting in decreases in intracellular H2O2 production and membrane upregulation of CD11b expression, both functions being inhibited after anti-NGF or anti-TrkA antibody treatment. K252a also inhibits PACAP-associated NF-KB activity. Monocytes increase in NGF production is seen after micromolar PACAP exposure while nanomolar treatment which desensitizes cells to high dose of PACAP prevents PACAP-induced TrkA phosphorylation, H2O2 production and CD11b expression. Finally, NGF-dependent ERK activation and H2O2 production is pertussis toxin sensitive. Altogether these data indicate that in PACAP-activated monocytes some pro-inflammatory activities occur through transactivation mechanisms involving VPAC-1, NGF and TrkA-associated tyrosine kinase activity.

Water exchange rates of water-soluble manganese(III) porphyrins of therapeutical potential.

The activation parameters and the rate constants of the water-exchange reactions of Mn(III)TE-2-PyP(5+) (meso-tetrakis(N-ethylpyridinium-2-yl)porphyrin) as cationic, Mn(III)TnHex-2-PyP(5+) (meso-tetrakis(N-n-hexylpyridinium-2-yl)porphyrin) as sterically shielded cationic, and Mn(III)TSPP(3-) (meso-tetrakis(4-sulfonatophenyl)porphyrin) as anionic manganese(iii) porphyrins were determined from the temperature dependence of (17)O NMR relaxation rates. The rate constants at 298 K were obtained as 4.12 x 10(6) s(-1), 5.73 x 10(6) s(-1), and 2.74 x 10(7) s(-1), respectively. On the basis of the determined entropies of activation, an interchange-dissociative mechanism (I(d)) was proposed for the cationic complexes (DeltaS(double dagger) = approximately 0 J mol(-1) K(-1)) whereas a limiting dissociative mechanism (D) was proposed for Mn(III)TSPP(3-) complex (DeltaS(double dagger) = +79 J mol(-1) K(-1)). The obtained water exchange rate of Mn(III)TSPP(3-) corresponded well to the previously assumed value used by Koenig et al. (S. H. Koenig, R. D. Brown and M. Spiller, Magn. Reson. Med., 1987, 4, 52-260) to simulate the (1)H NMRD curves, therefore the measured value supports the theory developed for explaining the anomalous relaxivity of Mn(III)TSPP(3-) complex. A magnitude of the obtained water-exchange rate constants further confirms the suggested inner sphere electron transfer mechanism for the reactions of the two positively charged Mn(iii) porphyrins with the various biologically important oxygen and nitrogen reactive species. Due to the high biological and clinical relevance of the reactions that occur at the metal site of the studied Mn(iii) porphyrins, the determination of water exchange rates advanced our insight into their efficacy and mechanism of action, and in turn should impact their further development for both diagnostic (imaging) and therapeutic purposes.

Functionally active targeting domain of the beta-adrenergic receptor kinase: an inhibitor of G beta gamma-mediated stimulation of type II adenylyl cyclase.

The beta-adrenergic receptor kinase (beta ARK) phosphorylates its membrane-associated receptor substrates, such as the beta-adrenergic receptor, triggering events leading to receptor desensitization. beta ARK activity is markedly stimulated by the isoprenylated beta gamma subunit complex of heterotrimeric guanine nucleotide-binding proteins (G beta gamma), which translocates the kinase to the plasma membrane and thereby targets it to its receptor substrate. The amino-terminal two-thirds of beta ARK1 composes the receptor recognition and catalytic domains, while the carboxyl third contains the G beta gamma binding sequences, the targeting domain. We prepared this domain as a recombinant His6 fusion protein from Escherichia coli and found that it had both independent secondary structure and functional activity. We demonstrated the inhibitory properties of this domain against G beta gamma activation of type II adenylyl cyclase both in a reconstituted system utilizing Sf9 insect cell membranes and in a permeabilized 293 human embryonic kidney cell system. Gi alpha-mediated inhibition of adenylyl cyclase was not affected. These data suggest that this His6 fusion protein derived from the carboxyl terminus of beta ARK1 provides a specific probe for defining G beta gamma-mediated processes and for studying the structural features of a G beta gamma-binding domain.

Phosphorylation/dephosphorylation of the beta-adrenergic receptor regulates its functional coupling to adenylate cyclase and subcellular distribution.

Prolonged exposure of cells or tissues to drugs or hormones such as catecholamines leads to a state of refractoriness to further stimulation by that agent, known as homologous desensitization. In the case of the beta-adrenergic receptor coupled to adenylate cyclase, this process has been shown to be intimately associated with the sequestration of the receptors from the cell surface through a cAMP-independent process. Recently, we have shown that homologous desensitization in the frog erythrocyte model system is also associated with increased phosphorylation of the beta-adrenergic receptor. We now provide evidence that the phosphorylation state of the beta-adrenergic receptor regulates its functional coupling to adenylate cyclase, subcellular translocation, and recycling to the cell surface during the process of agonist-induced homologous desensitization. Moreover, we show that the receptor phosphorylation is reversed by a phosphatase specifically associated with the sequestered subcellular compartment. At 23 degrees C, the time courses of beta-adrenergic receptor phosphorylation, sequestration, and adenylate cyclase desensitization are identical, occurring without a lag, exhibiting a t1/2 of 30 min, and reaching a maximum at approximately 3 hr. Upon cell lysis, the sequestered beta-adrenergic receptors can be partially recovered in a light membrane vesicle fraction that is separable from the plasma membranes by differential centrifugation. The increased beta-adrenergic receptor phosphorylation is apparently reversed in the sequestered vesicle fraction as the sequestered receptors exhibit a phosphate/receptor stoichiometry that is similar to that observed under basal conditions. High levels of a beta-adrenergic receptor phosphatase activity appear to be associated with the sequestered vesicle membranes. The functional activity of the phosphorylated beta-adrenergic receptor was examined by reconstituting purified receptor with its biochemical effector the guanine nucleotide regulatory protein (Ns) in phospholipid vesicles and assessing the receptor-stimulated GTPase activity of Ns. Compared to controls, phosphorylated beta-adrenergic receptors, purified from desensitized cells, were less efficacious in activating the Ns GTPase activity. These results suggest that phosphorylation of the beta-adrenergic receptor leads to its functional uncoupling and physical translocation away from the cell surface into a sequestered membrane domain. In the sequestered compartment, the phosphorylation is reversed thus enabling the receptor to recycle back to the cell surface and recouple with adenylate cyclase.

Beta-agonist- and prostaglandin E1-induced translocation of the beta-adrenergic receptor kinase: evidence that the kinase may act on multiple adenylate cyclase-coupled receptors.

beta-Adrenergic receptor kinase (beta-AR kinase) is a cytosolic enzyme that phosphorylates the beta-adrenergic receptor only when it is occupied by an agonist [Benovic, J. Strasser, R. H., Caron, M. G. & Lefkowitz, R. J. (1986) Proc. Natl. Acad. Sci. USA 83, 2797-2801.] It may be crucially involved in the processes that lead to homologous or agonist-specific desensitization of the receptor. Stimulation of DDT1MF-2 hamster smooth muscle cells or S49 mouse lymphoma cells with a beta-agonist leads to translocation of 80-90% of the beta-AR kinase activity from the cytosol to the plasma membrane. The translocation process is quite rapid, is concurrent with receptor phosphorylation, and precedes receptor desensitization and sequestration. It is also transient, since much of the activity returns to the cytosol as the receptors become sequestered. Stimulation of beta-AR kinase translocation is a receptor-mediated event, since the beta-antagonist propranolol blocks the effect of agonist. In the kin- mutant of the S49 cells (lacks cAMP-dependent protein kinase), prostaglandin E1, which provokes homologous desensitization of its own receptor, is at least as effective as isoproterenol in promoting beta-AR kinase translocation to the plasma membrane. However, in the DDT1MF-2 cells, which contain alpha 1-adrenergic receptors coupled to phosphatidylinositol turnover, the alpha 1-agonist phenylephrine is ineffective. These results suggest that the first step in homologous desensitization of the beta-adrenergic receptor may be an agonist-promoted translocation of beta-AR kinase from cytosol to plasma membrane and that beta-AR kinase may represent a more general adenylate cyclase-coupled receptor kinase that participates in regulating the function of many such receptors.

Un antigène soluble du virus de la mosaïque du tabac

SCOPUS: ar.j

Convenient synthetic routes to bidentate and monodentate 2-, 3- and 4-pyridyl phosphines: potentially useful ligands for water-soluble complex catalysts

The monodentate and bidentate pyridyl phosphines, PR3 and R2P(CH2)2PR2, where R=3- or 4-pyridyl can be prepared in high yields by treatment of butyllithium/TMEDA/3- or 4-bromopyridine with PCl3 or Cl2P(CH2)2PCl2 at low temperature. 1,2-Bis(di-2-pyridylphosphino)ethane is conveniently synthesised by an alternative route involving reaction of 1,2-dibromoethane with lithium di-2-pyridylphosphide.

Inclusion of poorly soluble drugs in highly ordered mesoporous silica nanoparticles

Silica nanoparticles (MSNs) with a highly ordered mesoporous structures (103A) with cubic Im3 m have been synthesized using triblock copolymers with high poly(alkylene oxide) (EO) segments in acid media. The produced nanoparticles displayed large specific surface area (approximately 765 cm(2)/g) with an average particles size of 120 nm. The loading efficiency was assessed by incorporating three major antiepileptic active substances via passive loading and it was found to varying from 17 to 25%. The state of the adsorbed active agents was further analyzed using differential scanning calorimetry (DSC) and X-ray powder diffraction (XRPD). Dissolution studies revealed rapid release profiles within the first 3 h. The viability of 3T3 endothelial cells was not affected in the presence of MSNs indicating negligible cytotoxicity. 2009 Elsevier B.V. All rights reserved.

In vitro drug release studies of polymeric freeze-dried wafers and solvent-cast films using paracetamol as a model soluble drug

Drug dissolution and release characteristics from freeze-dried wafers and solvent-cast films prepared from sodium carboxymethylcellulose (CMC) have been investigated to determine the mechanisms of drug release from the two systems. The formulations were prepared by freeze-drying (wafers) or drying in air (films), the hydrated gel of the polymer containing paracetamol as a model soluble drug. Scanning electron microscopy (SEM) was used to examine differences between the physical structure of the wafers and films. Dissolution studies were performed using an exchange cell and drug release was measured by UV spectroscopy at 242 nm. The effects of drug loading, polymer content and amount of glycerol (films) on the release characteristics of paracetamol were investigated. The release profiles of paracetamol from the wafers and films were also compared. A digital camera was used to observe the times to complete hydration and dissolution of the wafers containing different amounts of CMC and how that impacts on drug release rates. Both formulations showed sustained type drug release that was modelled by the Korsmeyer–Peppas equation. Changes in the concentration of drug and glycerol (films) did not significantly alter the rate of drug release while increasing polymer content significantly decreased the rate of drug release from both formulations. The results show that the rate of paracetamol release was faster from the wafers than the corresponding films due to differences in their physical structures. The wafers which formed a porous network, hydrated faster than the more dense and continuous, (non-porous) sheet-like structure of the films.