Surface immobilization of galactose onto aliphatic biodegradable polymers for hepatocyte culture

A novel surface modification method of biodegradable polymers was investigated for inducing the attachment of specific cells onto the polymer surface via ligand-receptor interactions. Galactose, a targeting ligand specific to asialoglycoprotein receptors present on cell membrane of hepatocytes, was introduced on the surface of poly(D,L-lactic-co-glycolic acid) (PLGA) films. A terminal end group of carboxylic acid in PLGA was activated by dicyclohexylcarbodiimide and N-hydroxysuccinimide for the direct conjugation of lactose by reductive amination reaction. Di-block copolymers of PLGA-b-poly(ethylene glycol) (PEG) having a free terminal amine group were also synthesized and used for the conjugation of galactose for the introduction of a PEG spacer between PLGA and galactose. The presence of galactose moieties on the blend film surface was characterized by measuring water contact angle and X-ray photon spectroscopy, and the amount of galactose was indirectly determined by a specific lectin-binding assay. With increasing the galactose concentration on the blend film surface, the initial attachment as well as the cell viability of hepatocyates concomitantly increased. The introduction of PEG spacer reduced the cell attachment and viability. Albumin secretion rate from hepatocytes was enhanced for galactose modified surfaces, whereas it was reduced for the surfaces not having galactose moieties.     

Hydrolysis of wheat starch and its effect on the falling number procedure: mathematical model

A population balance model was developed for wheat starch hydrolysis to simulate the performance parameters of a viscosity-based device, known as the Falling Number instrument. The instrument is widely used as an indirect means to gauge the level of preharvest sprout activity in cereal grains such as wheat and barley. The model consists of three competing kinetics: starch gelatinization, enzymatic hydrolysis, and enzyme thermal deactivation. Using established principles of starch rheology and fluid mechanics, the model simulates the velocity profiles of the falling stirrer, starch gel viscosity, and the Falling Number readings at various levels of alpha-amylase. Model predictions for the velocity of the stirrer at any time during the downward fall, as well as the prediction of the total time needed for the fall, defined as the Falling Number, were in fair agreement with experimental measurements. There was better agreement between the modeled viscosity and the final viscosity of the starch gel as measured by a precision rheometer than there was with the measured Falling Number.     

On the origin of modular variation

We study the dynamics of modularization in a minimal substrate. A module is a functional unit relatively separable from its surrounding structure. Although it is known that modularity is useful both for robustness and for evolvability (Wagner 1996), there is no quantitative model describing how such modularity might originally emerge. Here we suggest, using simple computer simulations, that modularity arises spontaneously in evolutionary systems in response to variation, and that the amount of modular separation is logarithmically proportional to the rate of variation. Consequently, we predict that modular architectures would appear in correlation with high environmental change rates. Because this quantitative model does not require any special substrate to occur, it may also shed light on the origin of modular variation in nature. This observed relationship also indicates that modular design is a generic phenomenon that might be applicable to other fields, such as engineering: Engineering design methods based on evolutionary simulation would benefit from evolving to variable, rather than stationary, fitness criteria, as a weak and problem-independent method for inducing modularity.     

Antimicrobial mechanism of β-glycyrrhetinic acid isolated from licorice, Glycyrrhiza glabra

-Glycyrrhetinic acid isolated from Glycyrrhiza glabra had an antibacterial activity of 7.6 and 12.5 g ml^–1 against Bacillus subtilis and Staphylococcus epidermidis without causing hemolysis of human erythrocytes, whereas it was not inhibitory against Escherichia coli, Proteus vulgaris and various fungi. Confocal microscopy showed that -glycyrrhetinic acid was located within the bacteria but had not caused membrane disruption. It then inhibited synthesis of DNA, RNA and protein.     

Halocidin: a new antimicrobial peptide from hemocytes of the solitary tunicate,Halocynthia aurantium

From hemocytes of the tunicate Halocynthia aurantium we purified a new antimicrobial peptide named halocidin. The native peptide had a mass of 3443 Da and comprised two different subunits containing 18 amino acid residues (WLNALLHHGLNCAKGVLA) and 15 residues (ALLHHGLNCAKGVLA), which were linked covalently by a single cystine disulfide bond. Two different monomers were separately synthesized and used to make three additional isoforms (15 residue homodimer, 18 residue homodimer, heterodimer). In antimicrobial assays performed with synthetic peptides of halocidin, it was confirmed that congeners of the 18 residue monomer were more active than those of the 15 residue monomer against methicillin-resistant Staphylococcus aureus and multidrug-resistant Pseudomonas aeruginosa.     

Multiple virus resistance in transgenic plants conferred by the human dsRNA-dependent protein kinase

We have developed a new strategy for engineering resistance to multipleviruses in plants. The strategy exploits the human double stranded (ds)RNA-dependent protein kinase (PKR). PKR is one of theinterferon-induced enzymes. It confers viral resistance in mammals byinhibitingviral replication through the inactivation of the translational initiationfactor, eIF-2, upon activation by dsRNA. The humanPKR gene was fused to the promoter of theArabidopsis blue copper binding protein gene(BCB) that is induced rapidly in response to wounding. Thechimeric gene cassette was introduced into tobacco plants. Expression of thePKR gene in transgenic tobacco plants was demonstrated byRNA gel blot analysis and autophosphorylation assay of anM _r 68,000 protein. The transgenic plantsexpressing the PKR gene showed significantly reduced viralsymptoms or no viral symptoms at all, when challenged by different plant RNAviruses, such as Cucumber mosaic virus, Tobaccoetch virus, or Potato virus Y. Thus, expressionof a single component in the human interferon pathway, thePKR gene, can effectively confer resistance to multipleviruses in transgenic plants.     

Processing and assembly in vitro of engineered soybean beta-conglycinin subunits with the asparagine-glycine proteolytic cleavage site of 11S globulins

A short interdomain sequence between the N- and C-terminal domains of beta-conglycinin, the major 7S seed storage protein of soybean, was selected as a target for insertion of amino acid residues specifically cleaved by an asparaginyl endopeptidase that processes globulins into acidic and basic chains. Modified beta-conglycinin subunits containing the proteolytic cleavage site self-assembled into trimers in vitro at an efficiency similar to that of the unmodified subunit. In contrast to the absence of cleavage of the unmodified subunits, however, the modified beta-conglycinin trimers were processed by purified soybean asparaginyl endopeptidase into two polypeptides, each the size expected for the beta-conglycinin N- and C-terminal domains, respectively. The cleavage did not alter the assembly of mutant beta-conglycinins and the cleaved mutant trimers remained stable to further proteolytic attack. To examine the possibility of coassembly between the cleaved 11S and 7S subunits, in vitro processed mutant beta-conglycinin subunits were mixed with native dissociated 11S globulin preparations. Reassembly at a high ionic condition did not induce the 7S subunits to interact with 11S subunits to form hexameric complexes. Thus, cleavage of 7S globulin subunits into acidic and basic domains may not be sufficient for hexamer assembly to occur. Biotechnological implications of the engineered proteins are discussed.     

Reprogramming of the activity of the activator/dissociation transposon family during plant regeneration in rice

Many aspects of epigenetic phenomena have been elucidated via studies of transposable elements. An active transposable element frequently loses its ability to mobilize and goes into an inactive state during development. In this study, we describe the cyclic activity of a maize transposable element dissociation (Ds) in rice. In rice genome, Ds undergoes the spontaneous loss of mobility. However, an inactive state of Ds can be changed into an active state during tissue culture. The recovery of mobility accompanies not only changes in the methylation patterns of the terminal region of Ds, but also alteration in the steady state level of the activator (Ac) mRNA that is expressed by a constitutive CaMV 35S promoter. Furthermore, the Ds-reactivation process is not random, but stage-specific during plantlet regeneration. Our findings have expanded previous observations on Ac reactivation in the tissue culture of maize.     

Secretory expression and purification of Aspergillus niger glucose oxidase in Saccharomyces cerevisiae mutant deficient in PMR1 gene

The gene encoding glucose oxidase (GOD) from Aspergillus niger was expressed as a secretory product in the yeast Saccharomyces cerevisiae. Six consecutive histidine residues were fused to the C-terminus of GOD to facilitate purification. The recombinant GOD-His(6) secreted by S. cerevisiae migrated as a broad diffuse band on SDS-PAGE, with an apparent molecular weight higher than that in natural A. niger GOD. To investigate the effects of hyperglycosylation on the secretion efficiency and enzyme properties, GOD-His(6) was expressed and secreted in a S. cerevisiae mutant in which the PMR1 gene encoding Ca(++)-ATPase was disrupted. The pmr1 null mutant strain secreted an amount of GOD-His(6) per unit cell mass higher than that in the wild-type strain. In contrast to the hyperglycosylated GOD-His(6) secreted in the wild-type strain, the pmr1 mutant strain secreted GOD-His(6) in a homogeneous form with a protein band pattern similar to that in natural A. niger GOD, based on SDS-PAGE. The hyperglycosylated and pmr1Delta mutant-derived GOD-His(6) enzymes were purified to homogeneity by immobilized metal ion-affinity chromatography and their specific activities and stabilities were compared. The specific activity of the pmr1Delta mutant-derived GOD-His(6) on a protein basis was very similar to that of the hyperglycosylated GOD-His(6), although its pH and thermal stabilities were lower than those of the hyperglycosylated GOD-His(6).     

Variation in mitochondrial DNA of Vietnamese pigs: relationships with Asian domestic pigs and Ryukyu wild boars

Mitochondrial DNA (mtDNA) sequences (574 bp) of 30 Vietnamese pigs (large and small) were examined and compared with those of 61 haplotypes from wild boars and domestic pigs from various locations in Asia. The large Vietnamese pigs had genetic links to Ryukyu wild boars in southern Japan. The small Vietnamese pigs were closely related to other East Asian domestic pigs. These results indicate that Vietnamese pigs are genetically diverse and may be descendents of wild and domestic pigs from other regions of Asia.