SummaryS-Adenosyl-L-methionine:(R,S)-reticuline 7-O-methyltransferase converts reticuline to laudanine in tetrahydrobenzylisoquinoline biosynthesis in the opium poppy Papaver somniferum. This enzyme activity has not yet been detected in plants. A proteomic analysis of P. somniferum latex identi®ed a gel spot that contained a protein(s) whose partial amino acid sequences were homologous to those of plant O-methyltransferases. cDNA was ampli®ed from P. somniferum RNA by reverse transcription PCR using primers based on these internal amino acid sequences. Recombinant protein was then expressed in Spodoptera frugiperda Sf9 cells in a baculovirus expression vector. Steady-state kinetic measurements with one heterologously expressed enzyme and mass spectrometric analysis of the enzymatic products suggested that this unusual enzyme is capable of carrying through sequential O-methylations on the isoquinoline and on the benzyl moiety of several substrates. The tetrahydrobenzylisoquinolines (R )-reticuline (4.2 sec À1 mM À1 ), (S )-reticuline (4.5 sec À1 mM À1 ), (R )-protosinomenine (1.7 sec À1 mM À1 ), and (R,S )-isoorientaline (1.4 sec À1 mM À1 ) as well as guaiacol (5.9 sec À1 mM À1 ) and isovanillic acid (1.2 sec À1 mM À1 ) are O-methylated by the enzyme with the ratio k cat /K m shown in parentheses. A P. somniferum cDNA encoding (R,S )-norcoclaurine 6-O-methyltransferase was similarly isolated and characterized. This enzyme was less permissive, methylating only (R,S )-norcoclaurine (7.4 sec À1 mM À1 ), (R )-norprotosinomenine (4.1 sec À1 mM À1 ), (S )-norprotosinomenine (4.0 sec À1 mM À1 ) and (R,S )-isoorientaline (1.0 sec À1 mM À1 ). A phylogenetic comparison of the amino acid sequences of these O-methyltransferases to those from 28 other plant species suggests that these enzymes group more closely to isoquinoline biosynthetic O-methyltransferases from Coptis japonica than to those from Thalictrum tuberosum that can O-methylate both alkaloid and phenylpropanoid substrates.
Delayed healing remains a major clinical problem and here we have sought to develop an improved dressing film comprising 1.95% w/v fibroin and 0.05% w/v aloe gel extract. The tensile strength of dry film was 21.1 ± 0.5 MPa and broke at 1.1 ± 0.2% elongation; corresponding values for wet film were 18.3 ± 1.3 MPa and 1.9 ± 0.1%. The film maintained its shape upon water immersion and the swelling ratio of the dry film was 0.8 ± 0.1 while the water uptake was 43.7 ± 2.6%. After 28 days of incubation in phosphate buffered saline (1 M, pH 7.4, 37 °C), the weight of film was reduced by 6.7 ± 1.1% and the tensile strength and elongation at breaking point (dry state) were 15.4 ± 0.6 MPa and 1.5 ± 0.2%, respectively. Compared to aloe-free fibroin film (2.0% fibroin extract only), the blended film enhanced the attachment and proliferation of skin fibroblasts. The bFGF immunofluorescence of fibroblasts cultured on the blended film appeared greater than those cultured on tissue culture plate or on aloe-free fibroin film while α-smooth muscle actin was maintained. In streptozotocin-induced diabetic rats, the wounds dressed with the blended film were smaller (p <0.05) by day 7 after wounding, compared to untreated diabetic wounds. Histology of repaired diabetic wounds showed the fibroblast distribution and collagen fiber organization to be similar to wounds in normal rats, and this was matched by enhanced hydroxyproline content. Thus, such accelerated wound healing by the blended fibroin/aloe gel films may find application in treatment of diabetic non-healing skin ulcers.
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Mucoadhesive polyethylenimine-dextran sulfate nanoparticles (PDNPs) were developed for local oral mucosa delivery. Punica granatum peel extract (PGE) was loaded into PDNPs for oral malodor reduction and caries prevention. PDNPs were constructed using the polyelectrolyte complexation technique employing oppositely charged polymers polyethylenimine (PEI) and dextran sulfate (DS), with PEG 400 as a stabilizer. Under optimal conditions, spherical particles of ∼ 500 nm with a zeta potential of ∼+28 mV were produced. Up to 98%, drug entrapment efficiency was observed. The mass ratio of PEI:DS played a significant role in controlling particle size and entrapment efficacy. PDNPs shown to be a good mucoadhesive drug delivery system as confirmed by ex vivo wash off test. In vitro dissolution studies revealed that PGE-loaded PDNPs manifested a prolong release characteristic with a burst release within 5 min. In addition, they remained effectively against oral bacteria.
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