Recycling of Overactivated Acyls by a Type II Thioesterase during Calcimycin Biosynthesis in Streptomyces chartreusis NRRL 3882.

Type II thioesterases typically function as editing enzymes, removing acyl groups that have been misconjugated to acyl carrier proteins during polyketide secondary metabolite biosynthesis as a consequence of biosynthetic errors. NRRL 3882 produces the pyrrole polyether ionophoric antibiotic, and we have identified the presence of a putative type II thioesterase-like sequence, , within the biosynthetic gene cluster involved in the antibiotic's synthesis. However, targeted gene mutagenesis experiments in which was inactivated in the organism did not lead to a decrease in calcimycin production but rather reduced the strain's production of its biosynthetic precursor, cezomycin.

Cezomycin Is Activated by CalC to Its Ester Form for Further Biosynthesis Steps in the Production of Calcimycin in Streptomyces chartreusis NRRL 3882.

Calcimycin, N-demethyl calcimycin, and cezomycin are polyether divalent cation ionophore secondary metabolites produced by A thorough understanding of the organization of their encoding genes, biosynthetic pathway(s), and cation specificities is vitally important for their efficient future production and therapeutic use. So far, this has been lacking, as has information concerning any biosynthetic relationships that may exist between calcimycin and cezomycin. In this study, we observed that when a Cal ( mutant) derivative of a calcimycin-producing strain of (NRRL 3882) was grown on cezomycin, calcimycin production was restored.

Quantum dot-antibody conjugates via carbodiimide-mediated coupling for cellular imaging.

This chapter describes the processes of antibody (Ab) production, purification, conjugation to quantum dots (QDs), and the use of the conjugates produced in intracellular imaging of cell components and structures. Specifically, information is provided on the conjugation of carboxyl surface-terminated QDs to Abs via a one-step reaction using the water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). The chapter details the process of conjugate optimization in terms of its final fluorescence and biological activity.

Surface engineering of nanoparticles in suspension for particle based bio-sensing.

Surface activation of nanoparticles in suspension using amino organosilane has been carried out via strict control of a particle surface ad-layer of water using a simple but efficient protocol 'Tri-phasic Reverse Emulsion' (TPRE). This approach produced thin and ordered layers of particle surface functional groups which allowed the efficient conjugation of biomolecules. When used in bio-sensing applications, the resultant conjugates were highly efficient in the hybrid capture of complementary oligonucleotides and the detection of food borne microorganism.

Headgroup effects on the Krafft temperatures and self-assembly of ω-hydroxy and ω-carboxy hexadecyl quaternary ammonium bromide bolaform amphiphiles: micelles versus molecular clusters?

Eight bolaform amphiphiles were synthesised and characterised; 4 α,ω-hydroxy-alkane trialkyl (and pyridyl) ammonium bromides and 4 α,ω-carboxy-alkane trialkyl (and pyridyl) ammonium bromides where the alkyl groups were methyl, ethyl and propyl. Four of these represented new compounds. Overall the Krafft temperatures (T(K)) of the eight amphiphiles were high, with 6 of the eight possessing T(K)s greater than 45 °C.

QD-antibody conjugates via carbodiimide-mediated coupling: a detailed study of the variables involved and a possible new mechanism for the coupling reaction under basic aqueous conditions.

A detailed study into the optimization of carbodiimide-mediated coupling of antibodies (Ab) and quantum dots (QD) for use in cellular imaging has been undertaken. This involved the grafting of commercially available carboxyl-modified QDs (Evident Technologies "Lake Placid Blue" Evitag and eBioscience's eflour nanocrystals) with anti-Cdc8 Abs to produce conjugates with specific affinity for fission yeast tropomyosin Cdc8 protein. The water-soluble carbodiimide 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) was used to activate the QDs prior to their incubation with antibody, and a range of QD-carboxyl/EDC/Ab mole ratios were used in the experiments in attempts to optimize fluorescence and bioaffinity of the conjugate products (EDC to QD-carboxyl-600 nmol/15 pmol to 0.

Silicon, silica and its surface patterning/activation with alkoxy- and amino-silanes for nanomedical applications.

Silica and silicates are widely used in nanomedicine with applications as diverse as medical device coatings to replacement materials in tissue engineering. Although much is known about silica and its synthesis, relatively few biomedical scientists fully appreciate the link that exists between its formulation and its resultant structure and function. This article attempts to provide insight into relevant issues in that context, as well as highlighting their importance in the material's eventual surface patterning/activation with alkoxy- and organo-silanes.

Encapsulation of superparamagnetic nanoparticles into red blood cells as new carriers of MRI contrast agents.

Aims: The half-life of superparamagnetic iron oxide nanoparticles in the bloodstream is very short since they are rapidly taken up by the reticuloendothelial system. In this article, we report the encapsulation of different magnetic nanoparticles into human erythrocytes to increase their blood circulation time.

Materials & Methods: Newly synthesized and commercially available nanoparticles were evaluated for the encapsulation into red blood cells through the transient opening of membrane pores by controlled hypotonic dialysis and successive isotonic resealing and reannealing of cells.

Novel and Improved Nanomaterials, Chemistries and Apparatus for Nanobiotechnology: the NACBO project.

This article outlines the nature and activities of the recently completed EU Framework Programme 6 Integrated Project, Novel and Improved Nanomaterials, Chemistries and Apparatus for Nanobiotechnology (NACBO). This project was designed to yield new nanomaterials, surface activation and synthetic nucleic acid chemistries, procedures and hardware for applications in forensics and diagnostics. It provides details on the project's structure and partnership along with its principal objectives and successes in terms of publications and commercial exploitation.

Design of water-based ferrofluids as contrast agents for magnetic resonance imaging.

We report the synthesis, characterization and relaxometric study of ferrofluids based on iron oxide, with potential for use as magnetic resonance imaging (MRI) contrast agents (CAs). The effect of different cost-effective, water-based surface modification approaches which can be easily scaled-up for the large scale synthesis of the ferrofluids has been investigated. Surface modification was achieved by silanization, and/or coating with non-toxic commercial dispersants (a lauric polysorbate and a block copolymer with pigment affinic groups, namely Tween 20 and Disperbyk 190) which were added after or during iron oxide nanoparticle synthesis.

Fabrication of novel hierarchically ordered porous magnetic nanocomposites for bio-catalysis.

Novel hierarchically ordered porous magnetic nanocomposites with interconnecting macroporous windows and meso-microporous walls containing well dispersed magnetic nanoparticles have been fabricated and used as a support to immobilise lipase for the efficient hydrolysis of ester.

Inorganic materials for bone repair or replacement applications.

In recent years, excipient systems have been used increasingly in biomedicine in reconstructive and replacement surgery, as bone cements, drug-delivery vehicles and contrast agents. Particularly, interest has been growing in the development and application of controlled pore inorganic ceramic materials for use in bone-replacement and bone-repair roles and, in this context, attention has been focused on calcium-phosphate, bioactive glasses and SiO2- and TiO2-based materials. It has been shown that inorganic materials that most closely mimic bone structure and surface chemistry most closely function best in bone replacement/repair and, in particular, if a substance possesses a macroporous structure (pores and interconnections >100 microm diameter), then cell infiltration, bone growth and vascularization can all be promoted.

Current molecular techniques for the detection of microbial pathogens.

Traditionally the detection of microbial pathogens in clinical, environmental or food samples has commonly needed the prelevation of cells by culture before the application ofthe detection strategy. This is done to increase cell number thereby overcoming problems associated with the sensitivity of classical detection strategies. However, culture-based methods have the disadvantages of taking longer, usually are more complex and require skilled personnel as well as not being able to detect viable but non cultivable microbial species.

Tn5530 from Burkholderia cepacia strain 2a encodes a chloride channel protein essential for the catabolism of 2,4-dichlorophenoxyacetic acid.

Chloride channel proteins (ClC) are found in living systems where they transport chloride ions across cell membranes. Recently, the structure/function of two prokaryotic ClC has been determined but little is known about the role of these proteins in the microbial metabolism of chlorinated compounds. Here we show that transposon Tn5530 from Burkholderia cepacia strain 2a encodes a ClC protein (BcClC) which is responsible for expelling Cl(-) ions generated during the catabolism of 2,4-dichlorophenoxyacetic acid (a chlorinated herbicide).

The use of magnetic nanoparticles in the development of new molecular detection systems.

Magnetic nanoparticles have been widely used in biomolecular separation and discrimination which coincidentally also represents the basis for most current day molecular diagnostic procedures. The specificity, affinity, and binding capacity of magnetic nanoparticles depends on their size, form, dispersion, and surface chemistry. In this review, we will briefly analyze how these factors affect biomolecular separations and focus on the use of magnetic nanoparticles in monitoring the microbial biodiversity in the environment.

Mesoporous silica-magnetite nanocomposite: fabrication and applications in magnetic bioseparations.

Magnetic bioseparations such as adsorption and elution of nucleic acids by a mesoporous superparamagnetic silica-magnetite nanocomposite are reported.

Surface modification of magnetic nanoparticles with alkoxysilanes and their application in magnetic bioseparations.

A versatile and inexpensive method for the introduction of amine groups onto the surface of silica-coated magnetite composite nanoparticles has been established based on the condensation of (aminopropyl)triethoxysilane (APTS). The process was observed to be sensitive to a range of variables, and a range of silane surface-modified nanoparticles was synthesized under various reaction conditions, that is, solvent systems [water, tetrahydrofuran (THF), ethanol, or 1:1 mixtures of them], reaction times (from 1 to 24 h), and temperatures (18, 50, and 70 degrees C), with water as the catalyst and silane at either 0.2% or 2% (w/v) in an attempt to optimize the process.