Rapid prototyping in tissue engineering: challenges and potential

Tissue engineering aims to produce patient-specific biological substitutes in an attempt to circumvent the limitations of existing clinical treatments for damaged tissue or organs. The main regenerative tissue engineering approach involves transplantation of cells onto scaffolds. The scaffold attempts to mimic the function of the natural extracellular matrix, providing a temporary template for the growth of target tissues. Scaffolds should have suitable architecture and strength to serve their intended function. This paper presents a comprehensive review of the fabrication methods, including conventional, mainly manual, techniques and advanced processing methods such as rapid prototyping (RP) techniques. The potential and challenges of scaffold-based technology are discussed from the perspective of RP technology.     

The binding characteristics and utilization of Aleuria aurantia,Lens culinaris and few other lectins in the elucidation of fucosyltransferase activities resembling cloned FT VI and apparently unique to colon cancer cells

Human colon carcinoma cell fucosyltransferase (FT) in contrast to the FTs of several human cancer cell lines, utilized GlcNAcbeta1,4GlcNAcbeta-O-Bn as an acceptor, the product being resistant to alpha1,6-L-Fucosidase and its formation being completely inhibited by LacNAc Type 2 acceptors. Further, this enzyme was twofold active towards the asialo agalacto glycopeptide as compared to the parent asialoglycopeptide. Only 60% of the GlcNAc moieties were released from [14C]fucosylated asialo agalacto triantennary glycopeptide by jack bean beta-N-acetylhexosaminidase. These alpha1,3-L-fucosylating activities on multiterminal GlcNAc residues and chitobiose were further examined by characterizing the products arising from fetuin triantennary and bovine IgG diantennary glycopeptides and their exoglycosidase-modified derivatives using lectin affinity chromatography. Utilization of [14C]fucosylated glycopeptides with cloned FTs indicated that Lens culinaris lectin and Aleuria aurantia lectin (AAL) required, respectively, the diantennary backbone and the chitobiose core alpha1,6-fucosyl residue for binding. The outer core alpha1,3- but not the alpha-1,2-fucosyl residues decreased the binding affinity of AAL. The AAL-binding fraction from [14C]fucosylated asialo fetuin, using colon carcinoma cell extract, contained 60% Endo F/PNGaseF resistant chains. Similarly AAL-binding species from [14C]fucosylated TFA-treated bovine IgG using colon carcinoma cell extract showed significant resistance to endo F/PNGaseF. However, no such resistance was found with the corresponding AAL non- and weak-binding species. Thus colon carcinoma cells have the capacity to fucosylate the chitobiose core in glycoproteins, and this alpha1,3-L-fucosylation is apparently responsible for the AAL binding of glycoproteins. A cloned FT VI was found to be very similar to this enzyme in acceptor substrate specificities. The colon cancer cell FT thus exhibits four catalytic roles, i.e., alpha1,3-L-fucosylation of: (a) Galbeta1,4GlcNAcbeta-; (b) multiterminal GlcNAc units in complex type chain; (c) the inner core chitobiose of glycopeptides and glycoproteins; and (d) the nonreducing terminal chiotobiose unit.     

Biosynthesis of the carbohydrate antigenic determinants, Globo H, blood group H, and Lewis b: a role for prostate cancer cell alpha1,2-L-fucosyltransferase

Prostate carcinoma LNCaP cells were unique among several human cancer cell lines which include two other prostate cancer cell lines, PC-3 and DU-145, in expressing alpha1,2-L-fucosyltransferase (FT) as an exclusive FT activity. Affinity gel-GDP and Sephacryl S100 HR columns were used for a partial purification of this enzyme from 3.9 x 10(9) LNCaP cells (approximately 200-fold; 40% yield). The K(m) value (2.7 mM) for the LacNAc type 2 acceptor was quite similar to the one reported for the cloned blood group H gene-specified alpha1,2-FT [Chandrasekaran et al. (1996) Biochemistry 35, 8914-8924]. N-Ethylmaleimide was a potent inhibitor (K(i ) 12.5 microM). The enzyme showed four-fold acceptor preference for the LacNAc type 2 unit in comparison to the T-hapten in mucin core 2 structure. Its main features were similar to those of the cloned enzyme: (1) C-6 sulfation of terminal Gal in the LacNAc unit increased the acceptor efficiency, whereas C-6 sialylation abolished acceptor ability; (2) C-6 sulfation of GlcNAc in LacNAc type 2 decreased by 80% the acceptor ability, whereas LacNAc type 1 was unaffected; (3) Lewis x did not serve as an acceptor; (4) the C-4 hydroxyl rather than the C-6 hydroxyl group of the GlcNAc moiety in LacNAc type1 was essential for activity; and (5) the acrylamide copolymer of Galbeta1,3GlcNAcbeta-O-Al was the best acceptor among the acrylamide copolymers. Additionally, highly significant biological features of alpha1,2FT were identified in the present study. The synthesis of Globo H and Lewis b determinants became evident from the fact that Galbeta1,3GalNAcbeta1,3Galalpha-O-Me and Galbeta1,3(Fucalpha1,4)Glc-NAcbeta1,3Galbeta-O-Me served as high-affinity acceptors for this enzyme. Further, D-Fucbeta1,3Gal-NAcbeta1,3Galalpha-O-Me was a very efficient acceptor, indicating that the C-6 hydroxyl group of the terminal Gal moiety in Globo H is not essential for the enzyme activity. Thus, the present study was able to demonstrate three different catalytic roles of LNCaP alpha1,2-FT, namely, the expressions of blood group H, Lewis b from Lewis a, and Globo H.     

Method for lipase-catalyzed carbonate synthesis via one- and two-step alkoxycarbonylation reactions

Lipase-catalyzed alkoxycarbonylation methods offer potential advantages over the currently practiced industrial scale chemical synthesis of carbonates. We report a method for synthesis of organic carbonates via lipase-catalyzed alkoxycarbonylation between diphenyl carbonate and various alcohols in hexane. This method utilizes precursors that are readily available and does not involve extensive purification of the intermediate. In a two-step process, the two phenyl groups of diphenyl carbonate were substituted by two alcohol nucleophiles. The approach was demonstrated for two-step synthesis of 14 different disubstituted carbonate products. The rates of reaction for the two steps were much slower if the order of nucleophile addition was reversed. Under optimal conditions, complete conversion of diphenyl carbonate occurred within 8-15 h at 50 degrees C, which is a significant improvement from 50-90 h at 24 degrees C. A kinetic model for the alkoxycarbonylation reaction was derived based on the Michaelis-Menten equation, which simplified to first-order kinetics at low and equimolar concentration of substrates.     

The Guanine Nucleotide Exchange Factor ARNO mediates the activation of ARF and phospholipase D by insulin

Background  

Phospholipase D (PLD) is involved in many signaling pathways. In most systems, the activity of PLD is primarily regulated by the members of the ADP-Ribosylation Factor (ARF) family of GTPases, but the mechanism of activation of PLD and ARF by extracellular signals has not been fully established. Here we tested the hypothesis that ARF-guanine nucleotide exchange factors (ARF-GEFs) of the cytohesin/ARNO family mediate the activation of ARF and PLD by insulin.     

Integrated control of fruit rot and powdery mildew of chilli using the biocontrol agent Pseudomonas fluorescens and a chemical fungicide

A combined strategy of chilli fruit rot and powdery mildew control consisting of reduced fungicide dose and biological control with antagonistic Pseudomonas fluorescens (Pf1) was evaluated. The sensitivity of P. fluorescens to fungicide azoxystrobin at different concentrations (100, 150, 200, 250 and 300 ppm) was tested by turbidometric method. The grown bacterium (Pf1) was tolerant to all concentrations of azoxystrobin. In two field trials, Pf1 tested in combination with reduced concentration of azoxystrobin was highly efficient in management of both diseases of chilli. Biological control of Colletotrichum capsici and Leveillula taurica with P. fluorescens (Pf1) was effective but less so than fungicide alone at the standard dose. However, combination of the biological control agent with a 50% reduction of fungicide dose was as effective as the standard fungicide alone. Application of P. fluorescens along with azoxystrobin significantly increased the survival of Pf1 in the phylloplane of chilli. Further, there were ‘twofold’ increases in activities of peroxidase, polyphenol oxidase, phenylalanine ammonia-lyase, β-1,3-glucanase, chitinase and phenolics in plants treated with Pf1 plus azoxystrobin.     

The molecular structure of human tissue type XV presents a unique conformation among the collagens

Establishing the structure of the non-fibrillar collagens has provided a unique perspective to understanding their specialized functions in the extracellular matrix. These proteins exhibit very diverse conformations and supramolecular assemblies. Type XV collagen is a large macromolecule distinguished by a highly interrupted collagenous domain and many utilized sites of attachment for CS (chondroitin sulfate) and HS (heparan sulfate) glycosaminoglycan chains. It is present in most basement membrane zones of human tissues, where it is found closely associated with large collagen fibrils. To determine the molecular shape and organization of type XV, the protein was purified from human umbilical cords by salt extraction, and by ion-exchange and antibody-affinity chromatography. The representation of type XV in one of its most abundant tissue sources is estimated at only (1-2)x10(-4)% of dry weight. The molecules examined by transmission electron microscopy after rotary shadowing were visualized in multiple forms. Relatively few type XV monomers appeared elongated and kinked; most molecules were found in a knot/figure-of-eight/pretzel configuration not previously described for a collagen. Collective measurements of these populations revealed an average length of 193+/-16 nm. At the N-terminal end, identified by C-terminal antibody binding, were three 7.7 nm-diameter spheres, corresponding to TSPN-1 (N-terminal module of thrombospondin-1) modules, and attached to the collagen backbone by a short linker. The type XV monomers show the ability to self-assemble into higher-order structures. Some were arranged in complex clusters, but simpler oligomers, which may represent intermediates, were observed in a cruciform pattern with intermolecular binding sites that probably originate in the interruption sequences. The morphology of type XV is thus the antithesis of the fibrillar collagens, and the shape attains the required flexibility to form the spectrum of interconnecting links between banded fibrils at the basement membrane/interstitial border. These type XV structures may act as a biological 'spring' to stabilize and enhance resilience to compressive and expansive forces, and the multimers, in particular, with selective complements of many localized CS and HS chains, may be instrumental in spatial and temporal recruitment of modulators in growth, development and pathological processes.