Effect of NaCl on biomass, protein and proline contents, and antioxidant enzymes in seedlings and calli of two Trigonella species

The effects of NaCl on growth, contents of proteins and proline, and activities of catalase, peroxidase and polyphenol oxidase were investigated in seedlings and calli of Trigonella foenum-graecum L. and T. aphanoneura Rech. f. Seeds and hypocotyl explants were cultured on Murashige and Skoog medium supplemented with 0, 50, 100, 150 and 200 mM NaCl. Seed germination and the fresh and dry mass of the seedlings decreased significantly under salinity. In both species significant increases in protein content of seedlings over that of control were observed at 150 and 200 mM NaCl. Protein content in calli decreased at 200 mM NaCl over that of control. Protein content was higher in seedlings than in calli at all NaCl concentrations. Conversely, proline content was lower in seedlings than in calli at all the tested NaCl concentrations. NaCl caused changes in the activities of peroxidase, catalase and polyphenol oxidase in seedlings and calli.     

Characterisation of capacitive field-effect sensors with a nanocrystalline-diamond film as transducer material for multi-parameter sensing

The feasibility of a capacitive field-effect EDIS (electrolyte-diamond-insulator-semiconductor) platform for multi-parameter sensing is demonstrated by realising EDIS sensors with an O-terminated nanocrystalline-diamond (NCD) film as transducer material for the detection of pH and penicillin concentration as well as for the label-free electrical monitoring of adsorption and binding of charged macromolecules, like polyelectrolytes. The NCD films were grown on p-Si-SiO(2) substrates by microwave plasma-enhanced chemical vapour deposition. To obtain O-terminated surfaces, the NCD films were treated in an oxidising medium. The NCD-based field-effect sensors have been characterised by means of constant-capacitance method. The average pH sensitivity of the O-terminated NCD film was 40 mV/pH. A low detection limit of 5 microM and a high penicillin G sensitivity of 65-70 mV/decade has been obtained for an EDIS penicillin biosensor with the adsorptively immobilised enzyme penicillinase. Alternating potential changes, having tendency to decrease with increasing the number of adsorbed polyelectrolyte layers, have been observed after the layer-by-layer deposition of polyelectrolyte multilayers, using positively charged PAH (poly (allylamine hydrochloride)) and a negatively charged PSS (poly (sodium 4-styrene sulfonate)) as a model system. The response mechanism of the developed EDIS sensors is discussed.     

Successful transduction of liver in hemophilia by AAV-Factor IX and limitations imposed by the host immune response

We have previously shown that a single portal vein infusion of a recombinant adeno-associated viral vector (rAAV) expressing canine Factor IX (F.IX) resulted in long-term expression of therapeutic levels of F.IX in dogs with severe hemophilia B. We carried out a phase 1/2 dose-escalation clinical study to extend this approach to humans with severe hemophilia B. rAAV-2 vector expressing human F.IX was infused through the hepatic artery into seven subjects. The data show that: (i) vector infusion at doses up to 2 x 10(12) vg/kg was not associated with acute or long-lasting toxicity; (ii) therapeutic levels of F.IX were achieved at the highest dose tested; (iii) duration of expression at therapeutic levels was limited to a period of approximately 8 weeks; (iv) a gradual decline in F.IX was accompanied by a transient asymptomatic elevation of liver transaminases that resolved without treatment. Further studies suggested that destruction of transduced hepatocytes by cell-mediated immunity targeting antigens of the AAV capsid caused both the decline in F.IX and the transient transaminitis. We conclude that rAAV-2 vectors can transduce human hepatocytes in vivo to result in therapeutically relevant levels of F.IX, but that future studies in humans may require immunomodulation to achieve long-term expression.     

Role of lysine-256 in Citrobacter freundii tyrosine phenol-lyase in monovalent cation activation

Tyrosine phenol-lyase (TPL) from Citrobacter freundii is dependent on monovalent cations, K(+) or NH(4)(+), for high activity. We have shown previously that Glu-69, which is a ligand to the bound cation, is important in monovalent cation binding and activation [Sundararaju, B., Chen, H., Shillcutt, S., and Phillips, R. S. (2000) Biochemistry 39, 8546-8555]. Lys-256 is located in the monovalent cation binding site of TPL, where it forms a hydrogen bond with a structural water bound to the cation. This lysine residue is highly conserved in sequences of TPL and the paralogue, tryptophan indole-lyase. We have now prepared K256A, K256H, K256R, and E69D/K256R mutant TPLs to probe the role of Lys-256 in monovalent cation binding and activation. K256A and K256H TPLs have low activity (k(cat)/K(m) values of 0.01-0.1%), are not activated by monovalent cations, and do not exhibit fluorescence emission at 500 nm from the PLP cofactor. In contrast, K256R TPL has higher activity (k(cat)/K(m) about 10% of wild-type TPL), is activated by K(+), and exhibits fluorescence emission from the PLP cofactor. K256A, K256H, and K256R TPLs bind PLP somewhat weaker than wild-type TPL. E69D/K256R TPL was prepared to determine if the guanidine side chain could substitute for the monovalent cation. This mutant TPL has wild-type activity with S-Et-L-Cys or S-(o-nitrophenyl)-L-Cys but has no detectable activity with L-Tyr. E69D/K256R TPL is not activated by monovalent cations and does not show PLP fluorescence. In contrast to wild-type and other mutant TPLs, PLP binding to E69D/K256R is very slow, requiring several hours of incubation to obtain 1 mol of PLP per subunit. Thus, E69D/K256R TPL appears to have altered dynamics. All of the mutant TPLs react with inhibitors, L-Ala, L-Met, and L-Phe, to form equilibrating mixtures of external aldimine and quinonoid intermediates. Thus, Lys-256 is not the base which removes the alpha-proton during catalysis. The results show that the function of Lys-256 in TPL is in monovalent cation binding and activation.     

A new thermostable α-l-arabinofuranosidase from a novel thermophilic bacterium

An alpha-L-arabinofuranosidase gene was identified in a sequenced genome of a novel thermophilic bacterium, which belongs to the recently described phylum of Thermomicrobia. Amino acid sequence comparison of the enzyme (designated AraF) revealed similarity to glycoside hydrolases of family 51. The gene was cloned into Escherichia coli and its recombinant product expressed and purified. The enzyme appeared to be a hexamer. AraF was optimally active at 70 degrees C (over 10 min) and pH 6 having 92% residual activity after 1 h at 70 degrees C. AraF had a Km) value of 0.6 mM and V(max) value of 122 U mg(-1) on p-nitrophenyl-alpha-L-arabinofuranoside. AraF was almost equally active on branched arabinan and debranched arabinan, properties not previously found in alpha-L-arabinofuranosidases in GH family 51.