A biological-inspired support frame for an artificial cornea

Bilateral corneal blindness represents a quarter of the total blind, world-wide. The artificial cornea in assorted forms, was developed to replace opaque non-functional corneas and to return sight in otherwise hopeless cases that were not amenable to corneal grafts; believed to be 2% of corneal blind. Despite technological advances in materials design and tissue engineering no artificial cornea has provided absolute, long-term success. Formidable problems exist, due to a combination of unpredictable wound healing and unmanageable pathology. To have a solid guarantee of reliable success an artificial cornea must possess three attributes: an optical window to replace the opaque cornea; a strong, long term union to surrounding ocular tissue; and the ability to induce desired host responses. A unique artificial cornea possesses all three functional attributes- the Osteo-odonto-keratoprosthesis (OOKP). The OOKP has a high success rate and can survive for up to twenty years, but it is complicated both in structure and in surgical procedure; it is expensive and not universally available. The aim of this project was to develop a synthetic substitute for the OOKP, based upon key features of the tooth and bone structure. In doing so, surgical complexity and biological complications would be reduced. Analysis of the biological effectiveness of the OOKP showed that the structure of bone was the most crucial component for implant retention. An experimental semi-rigid hydroxyapatite framework was fabricated with a complex bone-like architecture, which could be fused to the optical window. The first method for making such a framework, was pressing and sintering of hydroxyapatite powders; however, it was not possible to fabricate a void architecture with the correct sizes and uniformity of pores. Ceramers were synthesised using alternative pore forming methods, providing for improved mechanical properties and stronger attachment to the plastic optical window. Naturally occurring skeletal structures closely match the structural features of all forms of natural bone. Synthetic casts were fabricated using the replamineform process, of desirable natural artifacts, such as coral and sponges. The final method of construction by-passed ceramic fabrication in favour of pre-formed coral derivatives and focused on methods for polymer infiltration, adhesion and fabrication. Prototypes were constructed and evaluated; a fully penetrative synthetic OOKP analogue was fabricated according to the dimensions of the OOKP. Fabrication of the cornea shaped OOKP synthetic analogue was also attempted.

Investigation of the antiproliferative properties of tumour promoting phorbol esters and related compounds

Tumour promoting phorbol esters such as 12-0-tetradecanoylphorbol-13-acetate (TPA) exert a multitude of biological effects on many cellular systems, many of which are believed to be mediated via the activation of the enzyme protein kinase C (PKC). TPA and other biologically active phorbol esters inhibited the proliferation of the A549 human lung carcinoma cell line. However, after 5-6 days culture in the continued presence of the phorbol ester cells began to proliferate at a rate similar to that of untreated cells. Resistance to TPA was lost following subculturing, although subculture in the presence of 10 nM TPA for more than 9 weeks resulted in a more resistant phenotype. The selection of a TPA-resistant subpopulation was not responsible for the observed resistance. The antiproliferative properties of other PKC activators were investigated. Mezerein induced the same antiproliferative effects as TPA but synthetic diacylglycerols (DAGs), the presumed physiological ligands of PKC, exerted only a non-specific cytotoxic influence on growth. Bryostatins 1 and 2 were able to induce transient growth arrest of A549 cells in a manner similar to phorbol esters at nanomolar concentrations, but at higher concentrations blocked both their own antiproliferative action and also that of phorbol esters and mezerein. Fourteen compounds synthesized to mimic features of the phorbol ester pharmacophore and/or DAGs did not mimic the antiproliferative properties of TPA in A549 cells and exerted only a DAG-like non-specific cytotoxicity at high concentrations. The subcellular distribution and activity of PKC was determined following partial purification by non-denaturing polyacrylamide gel electrophoresis. Treatment with TPA, mezerein or bryostatins resulted in a concentration-dependent shift of PKC activity from the cytosol to cellular membranes within 30 min. Significant translocation was not observed on treatment with DAGs. Chronic exposure of cells to TPA caused a time- and concentration dependent down-regulation of functional PKC activity. A complete loss of PKC activity was also observed on treatment with growth-inhibitory concentrations of bryostatins. No PKC activity was detected in cells resistant to the growth-inhibitory influence of TPA. Measurement of intracellular Ca2+ concentrations using A549 cells cultured on Cytodex 1 microcarrier beads revealed that TPA, mezerein and the bryostatins induced a similar rapid rise in intracellular Ca2+ levels.

Biological barriers to the nasal delivery of peptide drugs

The nasal absorption of larger peptide and protein drugs is generally low. The importance of the mucus layer and enzymic degradation in reducing absorption were investigated. Reversed-phase high-performance liquid chromatographic (HPLC) methods were developed to assay a variety of compounds. Pig gastric mucus (PGM) was selected to investigate the importance of the mucus layer. A method of treating and storing PGM was developed and evaluated which was representative of the gel in vivo. The nature of the mucus barrier was evaluated in vitro with three-compartment diffusion cells and a series of compounds with differing physicochemical properties. Mucus retarded the diffusion of all the compounds with molecular weight and charge exerting a marked effect. Binding to mucus was investigated by a centrifugation method. All of the compounds tested were found to bind to mucus with the exception of the negatively charged molecule benzoic acid. The small peptides did not demonstrate greater binding to mucus than any of the other compounds evaluated. The effect of some absorption enhancers upon the rate of diffusion of tryptophan through mucus was determined in vi tro. At the concentrations employed the enhancers EDTA, N-acetylcysteine and taurodeoxycholic acid exerted no effect, whilst taurocholic acid and cholic acid, were found to slightly reduce the rate of diffusion. The intracellular and luminal proteolytic activity of the nose was investigated in the sheep animal model with a nasal mucosal homogenate and a nasal wash preparation respectively and a series of chemically similar peptides. Hydrolysis was also investigated with the proteolytic enzymes carboxypeptidase A, cytosolic leucine aminopeptidase and microsomal leucine aminopeptidase. Sheep nasal mucosa possesses significant peptide hydrolase activity capable of degrading all the substrates tested. Considerable variation in susceptibility was observed. Degradation occurred excl us i ve ly at the pept ide bond between the aromatic amino ac id and glycine, indicating some specificity for aromatic amino acids. Hydrolysis profiles indicated the presence of both aminopeptidase and carboxypeptidase enzymes. The specific activity of the microsomal fraction was found to be greater than the cytosolic fraction. Hydrolysis in the nasal wash indicated the presence of either luminal or loosely-bound proteases, which can degrade peptide substrates. The same specificity for aromatic amino acids was observed and aminopeptidase activity demonstrated. The specific activity of the nasal wash was smaller than that of the homogenate.

Gingipains from Porphyromonas gingivalis increase the chemotactic and respiratory burst-priming properties of the 77-amino-acid interleukin-8 variant

Porphyromonas gingivalis, a gram-negative anaerobe which is implicated in the etiology of active periodontitis, secretes degradative enzymes (gingipains) and sheds proinflammatory mediators (e.g., lipopolysaccharides [LPS]). LPS triggers the secretion of interleukin-8 (IL-8) from immune (72-amino-acid [aa] variant [IL-8(72aa)]) and nonimmune (IL-8(77aa)) cells. IL-8(77aa) has low chemotactic and respiratory burst-inducing activity but is susceptible to cleavage by gingipains. This study shows that both R- and K-gingipain treatments of IL-8(77aa) significantly enhance burst activation by fMLP and chemotactic activity (P < 0.05) but decrease burst activation and chemotactic activity of IL-8(72aa) toward neutrophil-like HL60 cells and primary neutrophils (P < 0.05). Using tandem mass spectrometry, we have demonstrated that R-gingipain cleaves 5- and 11-aa peptides from the N-terminal portion of IL-8(77aa) and the resultant peptides are biologically active, while K-gingipain removes an 8-aa N-terminal peptide yielding a 69-aa isoform of IL-8 that shows enhanced biological activity. During periodontitis, secreted gingipains may differentially affect neutrophil chemotaxis and activation in response to IL-8 according to the cellular source of the chemokine.

Biological interactions with synthetic polymers

Anchorage dependent cell culture is a useful model for investigating the interface that becomes established when a synthetic polymer is placed in contact with a biological system. The primary aim of this interdisciplinary study was to systematically investigate a number of properties that were already considered to have an influence on cell behaviour and thereby establish the extent of their importance. It is envisaged that investigations such as these will not only further the understanding of the mechanisms that affect cell adhesion but may ultimately lead to the development of improved biomaterials. In this study, surface analysis of materials was carried out in parallel with culture studies using fibroblast cells. Polarity, in it's ability to undergo hydrogen bonding (eg with water and proteins), had an important affect on cell behaviour, although structural arrangement and crystallinity were not found to exert any marked influence. In addition, the extent of oxidation that had occurred during the process of manufacture of substrates was also important. The treatment of polystyrene with a selected series of acids and gas plasmas confirmed the importance of polarity, structural groups and surface charge and it was shown that this polymer was not unique among `hydrophobic' materials in it's inability to support cell adhesion. The individual water structuring groups within hydrogel polymers were also observed to have controlling effects on cell behaviour. An overall view of the biological response to both hydrogel and non-hydrogel materials highlighted the importance of surface oxidation, polarity, water structuring groups and surface charge. Initial steps were also taken to analyse foetal calf serum, which is widely used to supplement cell culture media. Using an array of analytical techniques, further experiments were carried out to observe any possible differences in the amounts of lipids and calcium that become deposited to tissue culture and bacteriological grade plastic under cell culture conditions.

Signal processing for molecular and cellular biological physics:an emerging field

Recent advances in our ability to watch the molecular and cellular processes of life in action-such as atomic force microscopy, optical tweezers and Forster fluorescence resonance energy transfer-raise challenges for digital signal processing (DSP) of the resulting experimental data. This article explores the unique properties of such biophysical time series that set them apart from other signals, such as the prevalence of abrupt jumps and steps, multi-modal distributions and autocorrelated noise. It exposes the problems with classical linear DSP algorithms applied to this kind of data, and describes new nonlinear and non-Gaussian algorithms that are able to extract information that is of direct relevance to biological physicists. It is argued that these new methods applied in this context typify the nascent field of biophysical DSP. Practical experimental examples are supplied.

Studies of phospholipid oxidation by electrospray mass spectrometry: from analysis in cells to biological effects

The oxidation of lipids is important in many pathological conditions and lipid peroxidation products such as 4-hydroxynonenal (HNE) and other aldehydes are commonly measured as biomarkers of oxidative stress. However, it is often useful to complement this with analysis of the original oxidized phospholipid. Electrospray mass spectrometry (ESMS) provides an informative method for detecting oxidative alterations to phospholipids, and has been used to investigate oxidative damage to cells, and low-density lipoprotein, as well as for the analysis of oxidized phosphatidylcholines present in atherosclerotic plaque material. There is increasing evidence that intact oxidized phospholipids have biological effects; in particular, oxidation products of 1-palmitoyl-2-arachidonoyl-sn-glycerophosphocholine (PAPC) have been found to cause inflammatory responses, which could be potentially important in the progression of atherosclerosis. The effects of chlorohydrin derivatives of lipids have been much less studied, but it is clear that free fatty acid chlorohydrins and phosphatidylcholine chlorohydrins are toxic to cells at concentrations above 10 micromolar, a range comparable to that of HNE and oxidized PAPC. There is some evidence that chlorohydrins have biological effects that may be relevant to atherosclerosis, but further work is needed to elucidate their pro-inflammatory properties, and to understand the mechanisms and balance of biological effects that could result from oxidation of complex mixtures of lipids in a pathophysiological situation.

Transglutaminases:nature's biological glues

Transglutaminases (Tgases) are a widely distributed group of enzymes that catalyse the post-translational modification of proteins by the formation of isopeptide bonds. This occurs either through protein cross-linking via epsilon-(gamma-glutamyl)lysine bonds or through incorporation of primary amines at selected peptide-bound glutamine residues. The cross-linked products, often of high molecular mass, are highly resistant to mechanical challenge and proteolytic degradation, and their accumulation is found in a number of tissues and processes where such properties are important, including skin, hair, blood clotting and wound healing. However, deregulation of enzyme activity generally associated with major disruptions in cellular homoeostatic mechanisms has resulted in these enzymes contributing to a number of human diseases, including chronic neurodegeneration, neoplastic diseases, autoimmune diseases, diseases involving progressive tissue fibrosis and diseases related to the epidermis of the skin. In the present review we detail the structural and regulatory features important in mammalian Tgases, with particular focus on the ubiquitous type 2 tissue enzyme. Physiological roles and substrates are discussed with a view to increasing and understanding the pathogenesis of the diseases associated with transglutaminases. Moreover the ability of these enzymes to modify proteins and act as biological glues has not gone unnoticed by the commercial sector. As a consequence, we have included some of the present and future biotechnological applications of this increasingly important group of enzymes.

Receptor activity-modifying proteins 2 and 3 generate adrenomedullin receptor subtypes with distinct molecular properties

Adrenomedullin (AM) is a peptide hormone with numerous effects in the vascular systems. AM signals through the AM1 and AM2 receptors formed by the obligate heterodimerization of a G protein-coupled receptor, the calcitonin receptor-like receptor (CLR), and receptor activity-modifying proteins (RAMP) 2 and 3, respectively. These different CLR-RAMP interactions yield discrete receptor pharmacology and physiological effects. The effective design of therapeutics that target the individual AM receptors is dependent on understanding the molecular details of the effects of RAMPs on CLR. To understand the role of RAMPs 2 and 3 on the activation and conformation of the CLR subunit of AM receptors we mutated 68 individual amino acids in the juxtamembrane region of CLR, a key region for activation of AM receptors and determined the effects on cAMP signalling. Sixteen CLR mutations had differential effects between the AM1 and AM2 receptors. Accompanying this, independent molecular modelling of the full-length AM-bound AM1 and AM2 receptors predicted differences in the binding pocket, and differences in the electrostatic potential of the two AM receptors. Druggability analysis indicated unique features that could be used to develop selective small molecule ligands for each receptor. The interaction of RAMP2 or RAMP3 with CLR induces conformational variation in the juxtamembrane region, yielding distinct binding pockets, probably via an allosteric mechanism. These subtype-specific differences have implications for the design of therapeutics aimed at specific AM receptors and for understanding the mechanisms by which accessory proteins affect G protein-coupled receptor function.

Alternative exon usage in the single CPT1 gene of Drosophila generates functional diversity in the kinetic properties of the enzyme:differential expression of alternatively spliced variants in drosophila tissues

The Drosophila melanogaster genome contains only one CPT1 gene (Jackson, V. N., Cameron, J. M., Zammit, V. A., and Price, N. T. (1999) Biochem. J. 341, 483-489). We have now extended our original observation to all insect genomes that have been sequenced, suggesting that a single CPT1 gene is a universal feature of insect genomes. We hypothesized that insects may be able to generate kinetically distinct variants by alternative splicing of their single CPT1 gene. Analysis of the insect genomes revealed that (a) the single CPT1 gene in each and every insect genome contains two alternative exons and (ii) in all cases, the putative alternative splicing site occurs within a small region corresponding to 21 amino acid residues that are known to be essential for the binding of substrates and of malonyl-CoA in mammalian CPT1A.Weperformed PCR analyses of mRNA from different Drosophila tissues; both of the anticipated splice variants of CPT1mRNAwere found to be expressed in all of the tissues tested (both in larvae and adults), with the expression level for one of the splice variants being significantly different between flight muscle and the fat body of adult Drosophila. Heterologous expression of the full-length cDNAs corresponding to the two putative variants of Drosophila CPT1 in the yeast Pichia pastoris revealed two important differences between the properties of the two variants: (i) their affinity (K 0.5) for one of the substrates, palmitoyl-CoA, differed by 5-fold, and (ii) the sensitivity to inhibition by malonyl-CoA at fixed, higher palmitoyl-CoA concentrations was 2-fold different and associated with different kinetics of inhibition. These data indicate that alternative splicing that specifically affects a structurally crucial region of the protein is an important mechanism through which functional diversity of CPT1 kinetics is generated from the single gene that occurs in insects. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

Correlates of leaf optical properties in tropical forest sun and extreme-shade plants

Thirteens hade-adaptedr ain forest species were comparedw ith twelve sun-adaptedt ropical forest species for correlates to leaf optical properties (described previously in Amer. J. Bot. 73: 1100-1108). The two samples were similar in absorptance of quanta for photosynthesis, but the shade-adaptedt axa: 1) had significantlyl ower specificl eaf weights,i ndicatinga more metabolically efficient production of surface for quantum capture; 2) synthesized less chlorophyll per unit area; and 3) used less chlorophyll for capturing the same quanta for photosynthesis. The anatomical features that best correlate with this increased efficiency are palisade cell shape and chloroplast distribution. Palisade cells with more equal dimensions have more chloroplasts on their abaxial surfaces. This dense layer of chloroplasts maximizes the light capture efficiency limited by sieve effects. The more columnar palisade cells of sun-adapted taxa allow light to pass through the central vacuoles and spaces between cells, making chloroplasts less efficient in energy capture, but allowing light to reach chloroplasts in the spongy mesophyll. Pioneer species may be an exception to these two groups of species. Three pioneer taxa included in this study have columnar palisade cells that are extremely narrow and packed closely together. This layer allows little penetration of light, but exposure of the leaf undersurface may provide illumination of spongy mesophyll chloroplasts in these plants.

Leaf Optical Properties of Rainforest Sun and Extreme Shade Plants

The opticalp ropertieso f the leaves of twelve tropicals un speciesa nd thirteent ropicale xtreme shade species were examinedw ith an integratings pherea ttachedt o a spectroradiometerM. easurements of diffuse reflectance and transmittance allowed calculations of absorptance, 350- 1,100 nm. Althoughs ome shade species absorbedh igherp ercentageso f quantumf lux densities for photosynthesis (400-700 nm, PPFD) than the mean for the sun species, the sun and shade species as groups were not significantly different from each other: 90.2, S.D. 3.6% for shade species and 88.6, S.D. 2.4% for the sun species. The groups of species did not differ in total absorptance of energy 350-1,100 nm. Furthermore, the sun and shade species were identical in theirs hifto f absorptancea t wavelengthsb etween6 50 and 750 nm. The anthocyanicc oloration of the leaf undersurfaceso f two species polymorphicf or this characteristic( Trionela hirsuta and Ischnosciphonp ruinosus)i s correlatedw ith increaseda bsorptancea t the uppere nd of the action spectrum of photosynthesis. Although sun and shade species have similar optical properties, the energy investment (as documented by dry wt per unit area of leaf surface) is much less for the shade species.

Synthesis and Biological Evaluation of Novel Folic Acid Receptor-Targeted, β-Cyclodextrin-Based Drug Complexes for Cancer Treatment

Drug targeting is an active area of research and nano-scaled drug delivery systems hold tremendous potential for the treatment of neoplasms. In this study, a novel cyclodextrin (CD)-based nanoparticle drug delivery system has been assembled and characterized for the therapy of folate receptor-positive [FR(+)] cancer. Water-soluble folic acid (FA)-conjugated CD carriers (FACDs) were successfully synthesized and their structures were confirmed by 1D/2D nuclear magnetic resonance (NMR), matrix-assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF-MS), high performance liquid chromatography (HPLC), Fourier transform infrared spectroscopy (FTIR), and circular dichroism. Drug complexes of adamatane (Ada) and cytotoxic doxorubicin (Dox) with FACD were readily obtained by mixed solvent precipitation. The average size of FACD-Ada-Dox was 1.5–2.5 nm. The host-guest association constant Ka was 1,639 M−1 as determined by induced circular dichroism and the hydrophilicity of the FACDs was greatly enhanced compared to unmodified CD. Cellular uptake and FR binding competitive experiments demonstrated an efficient and preferentially targeted delivery of Dox into FR-positive tumor cells and a sustained drug release profile was seen in vitro. The delivery of Dox into FR(+) cancer cells via endocytosis was observed by confocal microscopy and drug uptake of the targeted nanoparticles was 8-fold greater than that of non-targeted drug complexes. Our docking results suggest that FA, FACD and FACD-Ada-Dox could bind human hedgehog interacting protein that contains a FR domain. Mouse cardiomyocytes as well as fibroblast treated with FACD-Ada-Dox had significantly lower levels of reactive oxygen species, with increased content of glutathione and glutathione peroxidase activity, indicating a reduced potential for Dox-induced cardiotoxicity. These results indicate that the targeted drug complex possesses high drug association and sustained drug release properties with good biocompatibility and physiological stability. The novel FA-conjugated β-CD based drug complex might be promising as an anti-tumor treatment for FR(+) cancer.

Site specific growth of metal catalyzed silica nanowires for biological and chemical sensing

In this research the integration of nanostructures and micro-scale devices was investigated using silica nanowires to develop a simple yet robust nanomanufacturing technique for improving the detection parameters of chemical and biological sensors. This has been achieved with the use of a dielectric barrier layer, to restrict nanowire growth to site-specific locations which has removed the need for post growth processing, by making it possible to place nanostructures on pre-pattern substrates. Nanowires were synthesized using the Vapor-Liquid-Solid growth method. Process parameters (temperature and time) and manufacturing aspects (structural integrity and biocompatibility) were investigated. Silica nanowires were observed experimentally to determine how their physical and chemical properties could be tuned for integration into existing sensing structures. Growth kinetic experiments performed using gold and palladium catalysts at 1050°C for 60 minutes in an open-tube furnace yielded dense and consistent silica nanowire growth. This consistent growth led to the development of growth model fitting, through use of the Maximum Likelihood Estimation (MLE) and Bayesian hierarchical modeling. Transmission electron microscopy studies revealed the nanowires to be amorphous and X-ray diffraction confirmed the composition to be SiO2 . Silica nanowires were monitored in epithelial breast cancer media using Impedance spectroscopy, to test biocompatibility, due to potential in vivo use as a diagnostic aid. It was found that palladium catalyzed silica nanowires were toxic to breast cancer cells, however, nanowires were inert at 1μg/mL concentrations. Additionally a method for direct nanowire integration was developed that allowed for silica nanowires to be grown directly into interdigitated sensing structures. This technique eliminates the need for physical nanowire transfer thus preserving nanowire structure and performance integrity and further reduces fabrication cost. Successful nanowire integration was physically verified using Scanning electron microscopy and confirmed electrically using Electrochemical Impedance Spectroscopy of immobilized Prostate Specific Antigens (PSA). The experiments performed above serve as a guideline to addressing the metallurgic challenges in nanoscale integration of materials with varying composition and to understanding the effects of nanomaterials on biological structures that come in contact with the human body.