A thermoresponsive chitosan-NIPAAm/vinyl laurate copolymer vector for gene transfection

A carboxyl-terminated N-isopropylacrylamide/vinyl laurate (VL) copolymer was prepared and coupled with chitosan (molecular weight = 2000) to produce a chitosan-NIPAAm/VL copolymer (PNVLCS) vector. The aqueous solution of PNVLCS displayed an obvious thermoresponsive behavior with a lower critical solution temperature (LCST) about 26 degrees C. The transmission electron microscopy (TEM) showed that the size of PNVLCS/DNA complexes varied with charge ratios (+/-), and the smaller nanoparticles were formed at higher charge ratios. DLS revealed that the size of complex particles was dependent on temperature. The results of temperature-variable circular dichroism (CD), UV, and electrophoresis retardation indicated that at lower charge ratios, DNA in the complexes assume a B conformation, whereas increasing charge ratios caused B --> C type conformation transformation; the dissociation-formation of PNVLCS/DNA complexes could be tuned by varying temperature: at 37 degrees C, the collapse of PNIPAAm in PNVLCS was favorable for the formation of compact complexes, shielding more DNA from exposure; at 20 degrees C, the hydrated and extended PNIPAAm chains facilitated the unpacking of DNA from PNVLCS, increasing the exposure of DNA. PNVLCS was used to transfer plasmid-encoding beta-galactosidase into C2C12 cells. The level of gene expression could be controlled by varying incubation temperature. The transfection efficiency of PNVLCS was well improved by temporarily reducing culture temperature to 20 degrees C, whereas naked DNA and Lipofectamine 2000 did not demonstrate the characteristics of thermoresponsive gene transfection.     

Use of Ssp dnaB derived mini-intein as a fusion partner for production of recombinant human brain natriuretic peptide in Escherichia coli

To prevent in vivo degradation, small peptides are usually expressed in fusion proteins from which target peptides can be released by proteolytic or chemical reagents. In this report, a modified Ssp dnaB mini-intein linked with a chitin binding domain tag was used as a fusion partner for production of human brain natriuretic peptide (hBNP), a hormone for the treatment of congestive heart failure. The fusion protein was expressed as an inclusion body in Escherichia coli. After refolding, the fusion protein was purified with a chitin affinity column, and dnaB mini-intein mediated peptide-bond hydrolysis was triggered by shifting the pH in the chitin column to 7.0 at 25 degrees C for 16 h, which led to the release and separation of hBNP from its fusion partner. The hBNP sample was further purified with reverse phase HPLC and its biological activity was assayed in vitro. It was found that hBNP had a potent vasodilatory effect on rabbit aortic strips with an EC(50) of (1.24+/-0.32)x10(-6)mg/ml, which was similar to that of the synthetic BNP standard. The expression strategy described here promises to produce small peptides without use of proteolytic or chemical reagents.     

Fringe glycosyltransferases differentially modulate Notch1 proteolysis induced by Delta1 and Jagged1

Fringe O-fucose-beta1,3-N-acetylglucosaminyltransferases modulate Notch signaling by potentiating signaling induced by Delta-like ligands, while inhibiting signaling induced by Serrate/Jagged1 ligands. Based on binding studies, the differential effects of Drosophila fringe (DFng) on Notch signaling are thought to result from alterations in Notch glycosylation that enhance binding of Delta to Notch but reduce Serrate binding. Here, we report that expression of mammalian fringe proteins (Lunatic [LFng], Manic [MFng], or Radical [RFng] Fringe) increased Delta1 binding and activation of Notch1 signaling in 293T and NIH 3T3 cells. Although Jagged1-induced signaling was suppressed by LFng and MFng, RFng enhanced signaling induced by either Delta1 or Jagged1, underscoring the diversity of mammalian fringe glycosyltransferases in regulating signaling downstream of different ligand-receptor combinations. Interestingly, suppression of Jagged1-induced Notch1 signaling did not correlate with changes in Jagged1 binding as found for Delta1. Our data support the idea that fringe glycosylation increases Delta1 binding to potentiate signaling, but we propose that although fringe glycosylation does not reduce Jagged1 binding to Notch1, the resultant ligand-receptor interactions do not effectively promote Notch1 proteolysis required for activation of downstream signaling events.     

Identification and molecular characterization of the rainbow trout calpains (Capn1 and Capn2): their expression in muscle wasting during starvation

Calpains are calcium regulated proteases involved in cellular functions that include muscle proteolysis both ante- and postmortem. Here, we describe the molecular characterization of the rainbow trout catalytic subunits of the mu- and m-calpains, respectively. The cDNA sequence for Capn1 encodes a protein of 704 amino acids with a calculated molecular mass of 79.9 kDa. The amino acid sequence shows 66% and 86% identity with the mouse and zebrafish Capn1, respectively. The Capn2 cDNA codes for a protein consisting of 701 amino acid residues with a calculated molecular mass of 78.2 kDa. The protein shows 65% amino acid sequence identity with the mouse and chicken Capn2. The two isozymes of rainbow trout have the characteristic domains: I (propeptide), II (cysteine catalytic site), III (electrostatic switch), and IV (contains five EF-hands). Because starvation induces muscle wasting, the hypothesis of this study was that starvation could affect regulation of the calpain system in muscle. Starvation of rainbow trout fingerlings (15-20 g) for 35 days stimulated the expression of Capn1 (2.2-fold increase, P < 0.01), Capn2 (6.0-fold increase, P < 0.01), and calpastatins (1.6-fold increase, P < 0.05) as measured by quantitative real-time RT-PCR. The mRNA changes led to a 1.23-fold increase in the calpain catalytic activity. The results suggest a potential role of calpains in protein mobilization as a source of energy under fasting condition.     

Effects of a biocontrol agent and methyl jasmonate on postharvest diseases of peach fruit and the possible mechanisms involved

AIMS: To investigate effects of application of 200 micromol l(-1) methyl jasmonate [MeJA (200)] and Cryptococcus laurentii alone or in combination against postharvest diseases (Monilinia fructicola and Penicillium expansum) in peach fruit stored at 25 and 0 degrees C, and to evaluate the possible mechanisms involved. METHODS AND RESULTS: The efficacy of controlling postharvest diseases by resistance induced in peach fruit treated with MeJA (200) and C. laurentii alone or in combination and the relationship between activities of defence-related enzymes in peach fruit and lesions caused by M. fructicola and P. expansum were examined. At the same time, the effects of MeJA (200) on the population of C. laurentii in the peach wounds and on the mycelial growth of M. fructicola and P. expansumin vitro were investigated. The results indicated that treatment of peach fruit with C. laurentii at 1 x 10(8) CFU ml(-1) alone, or combining C. laurentii at 5 x 10(7) CFU ml(-1) with MeJA (200) all resulted in a lower lesion diameter of brown rot and blue mould caused by M. fructicola and P. expansum compared with the controls in peach fruit. MeJA (200) enhanced the population of C. laurentii, and inhibited mycelial growth of P. expansum. However, it had a little effect on M. fructicolain vitro. MeJA and C. laurentii alone or in combination induced higher activities of Chitinase, beta-1,3-glucanase, phenylalanine ammonia-lyase and peroxidase (POD) than applying the yeast alone at both 25 and 0 degrees C. CONCLUSIONS: MeJA (200) not only directly inhibited mycelial spread of postharvest pathogens, but also increased population of C. laurentii, which induced stronger disease resistance in fruit than MeJA or yeast alone, and resulted in a lower lesion diameter of brown rot and blue mould caused by M. fructicola and P. expansum. SIGNIFICANCE AND IMPACT OF THE STUDY: MeJA (200) in combination with C. laurentii was beneficial for controlling brown rot and blue mould caused by M. fructicola and P. expansum in peach fruit. The inhibitory mechanism was mainly because of resistance induced in peach fruit by MeJA and C. laurentii. In addition, direct inhibition of MeJA on P. expansum also played a role in controlling blue mould.     

High level expression of a recombinant acid phytase gene in Pichia pastoris

AIMS: To achieve high phytase yield with improved enzymatic activity in Pichia pastoris. METHODS AND RESULTS: The 1347-bp phytase gene of Aspergillus niger SK-57 was synthesized using a successive polymerase chain reaction and was altered by deleting intronic sequences, optimizing codon usage and replacing its original signal sequence with a synthetic signal peptide (designated MF4I) that is a codon-modified Saccharomyces cerevisiae mating factor alpha-prepro-leader sequence. The gene constructs containing wild type or modified phytase gene coding sequences under the control of the highly-inducible alcohol oxidase gene promoter with the MF4I- or wild type alpha-signal sequence were used to transform Pichia pastoris. The P. pastoris strain that expressed the modified phytase gene (phyA-sh) with MF4I sequence produced 6.1 g purified phytase per litre of culture fluid, with the phytase activity of 865 U ml(-1). The expressed phytase varied in size (64, 67, 87, 110 and 120 kDa), but could be deglycosylated to produce a homogeneous 64 kDa protein. The recombinant phytase had two pH optima (pH 2.5 and pH 5.5) and an optimum temperature of 60 degrees C. CONCLUSIONS: The P. pastoris strain with the genetically engineered phytase gene produced 6.1 g l(-1) of phytase or 865 U ml(-1) phytase activity, a 14.5-fold increase compared with the P. pastoris strain with the wild type phytase gene. SIGNIFICANCE AND IMPACT OF THE STUDY: The P. pastoris strain expressing the modified phytase gene with the MF4I signal peptide showed great potential as a commercial phytase production system.     

Matching base-pair number dependence of the kinetics of DNA-DNA hybridization studied by surface plasmon fluorescence spectroscopy

Two single-stranded DNA oligonucleotides consisting of complementary base-pairs can form double strands. This phenomenon is well studied in solutions, however, in order to clarify the physical mechanism of the hybridization occurring at a solid/solution interface, we studied the kinetics by surface plasmon fluorescence spectroscopy (SPFS): one single-stranded oligo-DNA (probe-DNA) was immobilized on the substrate, the other one (target-DNA) labelled with a fluorescent probe was added to the flow cell. After hybridization, the chromophores could be excited by the surface plasmon mode and their fluorescence detected with high sensitivity. The dependence of the k(on) and k(off) rate constants on the length of the hybridizing oligonucleotides was investigated by using a MM0 series (no mismatch) and the kinetics was found to be well described by a Langmuir adsorption model. From these measurements we found that also in the case of surface hybridization the affinity of the duplexes decreases as the number of matching base-pairs decreases from 15 to 10. In order to show that SPFS is the powerful technique with high sensitivity, the hybridization process for mixed target-oligos was measured by SPFS and analyzed by an expanded Langmuir model in which two components of target-oligo can bind to probe-DNA at the sensor surface competitively. Two sets of the k(on) and k(off) obtained from the experiment are successfully consistent with the k(on) and k(off) obtained from experiments for single (pure) target-DNA.     

Site-specific immobilization of proteins in a microarray using intein-mediated protein splicing

One of the critical issues in the generation of a protein microarray lies in the choice of immobilization strategies, which ensure proteins are adhered to the glass surface while properly retaining their native biological activities. Herein, we report a bacterium-based, intein-mediated strategy to generate N-terminal cysteine-containing proteins which are then chemoselectively immobilized to a thioester-functionalized glass slide to generate the corresponding protein microarray. We also showed preliminary data of the strategy in a yeast host system.     

Synthesis of alkyne derivatives of a novel triazolopyrazine as A(2A) adenosine receptor antagonists

A novel [1,2,4]triazolo[1,5-a]pyrazine core was synthesized and coupled with terminal acetylenes. The structure-activity relationship of the alkynes from this novel template was studied for their in vitro and in vivo adenosine A(2A) receptor antagonism. Selected compounds from this series were shown to have potent in vitro and in vivo activities against adenosine A(2A) receptor. Compound 12, in particular, was found to be orally active at 3mg/kg in both a mouse catalepsy model and a 6-hydroxydopamine-lesioned rat model.