An extracellular transglutaminase is required for apple pollen tube growth

An extracellular form of the calcium-dependent protein-cross-linking enzyme TGase (transglutaminase) was demonstrated to be involved in the apical growth of Malus domestica pollen tube. Apple pollen TGase and its substrates were co-localized within aggregates on the pollen tube surface, as determined by indirect immunofluorescence staining and the in situ cross-linking of fluorescently labelled substrates. TGase-specific inhibitors and an anti-TGase monoclonal antibody blocked pollen tube growth, whereas incorporation of a recombinant fluorescent mammalian TGase substrate (histidine-tagged green fluorescent protein: His6-Xpr-GFP) into the growing tube wall enhanced tube length and germination, consistent with a role of TGase as a modulator of cell wall building and strengthening. The secreted pollen TGase catalysed the cross-linking of both PAs (polyamines) into proteins (released by the pollen tube) and His6-Xpr-GFP into endogenous or exogenously added substrates. A similar distribution of TGase activity was observed in planta on pollen tubes germinating inside the style, consistent with a possible additional role for TGase in the interaction between the pollen tube and the style during fertilization.

Characterization of FITC-conjugated lectin binding to Candida albicans

Avidity of yeast and hyphal forms of Candida albicans for FITC-conjugated lectins was determined by flow cytometry and digital microscopy. Yeast phase cells bound Con A, a lectin with marked affinity for mannose, irrespective of growth phase, yet demonstrated little avidity for WGA and SBA. Yeast phase cell avidity for mannose-specific lectins was characterized through determination of FITC-conjugated Con A, LcH, PSA and GNA binding and subsequent calculation of Bmax, EC50 and Hn values. Such an approach, through comparison among FITC-conjugated lectins of differing specific activities, furnishes further insight into exposed outer cell wall mannose moieties. The rank order of lectin affinity as defined by EC50 values was GNA > Con A > LcH > PSA. Values for Hn suggest that lectins predominantly bind to a single receptor class, the relative abundance of which as defined by Bmax values was PSA > GNA > Con A > LcH. Hyphal surfaces in common with yeast phase cells demonstrated marked avidity for FITC-Con A, however, fluorescence of Candida morphological forms differed significantly, indicative of varying outer cell wall mannose exposure.

Variation in Miscanthus chemical composition and implications for conversion by pyrolysis and thermo-chemical bio-refining for fuels and chemicals

Different species and genotypes of Miscanthus were analysed to determine the influence of genotypic variation and harvest time on cell wall composition and the products which may be refined via pyrolysis. Wet chemical, thermo-gravimetric (TGA) and pyrolysis-gas chromatography–mass spectrometry (Py-GC–MS) methods were used to identify the main pyrolysis products and determine the extent to which genotypic differences in cell wall composition influence the range and yield of pyrolysis products. Significant genotypic variation in composition was identified between species and genotypes, and a clear relationship was observed between the biomass composition, yields of pyrolysis products, and the composition of the volatile fraction. Results indicated that genotypes other than the commercially cultivated Miscanthus x giganteus may have greater potential for use in bio-refining of fuels and chemicals and several genotypes were identified as excellent candidates for the generation of genetic mapping families and the breeding of new genotypes with improved conversion quality characteristics.

Osmotic dehydration in plant tissues

The primary aim of the thesis is to provide a comprehensive investigation of the osmotic dehydration processes in plant tissue. Effort has been concentrated on the modelling for simulating the processes. Two mathematical models for simulating the mass transfer during osmotic dehydration processes in plant tissues are developed and verified using existing experimental data. Both models are based on the mechanism of diffusion and convection of any mobile material that can transport in plant tissues. The mass balance equation for the transport of each constituent is established separately for intracellular and extra-cellular volumes with taking into account the mass transfer across the cell membrane the intracellular and extra-cellular volumes and the shrinkage of the whole tissue. The contribution from turgor pressure is considered in both models. Model two uses Darcy’s law to build the relation between shrinkage velocity and hydrostatic pressure in each volume because the plant tissue can be considered as the porous medium. Moreover, it has been extended to solve the multi-dimensional problems. A lot of efforts have been made to the parameter study and the sensitivity analyses. The parameters investigated including the concentration of the osmotic solution, diffusion coefficient, permeability of the cell membrane, elastic modulus of the cell wall, critical cell volume etc. The models allow us to quantitatively simulate the time evolution of intracellular and extra-cellular volumes as well as the time evolution of concentrations in each cross-section.

Mediators of gram-positive septic shock

Septic shock can occur as a result of Gram-negative or Gram-positive infection and involves a complex interaction between bacterial factors and the host immune system producing a systemic inflammatory state that may progress to multiple organ failure and death. Gram-positive bacteria are increasingly becoming more prevalent especially Staphylococcus epidermidis in association with indwelling devices. Lipopolysaccaride (LPS) is the key Gram-negative component involved in this process, but it is not clear which components of Gram-positive bacteria are responsible for progression of this often fatal disease. The aim of this thesis was to investigate the effect of bacterial components on the immune systems. Lipid S, a short chain form of lipoteichoic acid (LTA) found to be excreted from bacteria during growth in culture medium was examined along with other Gram-positive cell wall components: LTA, peptidoglycan (PG) and wall teichoic acids (WTA) and LPS from Gram-negative bacteria. Lipid S, LTA, PG and LPS but not WTA all stimulated murine macrophages and cell lines to produce significant amounts of NO, TNF-a, IL-6 and IL-1 and would induce fever and tissue damage seen in inflammatory diseases. Lipid S proved to be the most potent out of the Gram-positive samples tested. IgG antibodies in patients serum were found to bind to and cross react with lipid S and LTA. Anti-inflammatory antibiotics, platelet activating factor (PAF), PAF receptor antagonists and monoclonal antibodies (mAbs) directed to LTA, CD14 and toll-like receptors were utilised to modulate cytokine and NO production. In cell culture the anti-LTA and the anti-CD14 mAbs failed to markedly attenuate the production of NO, TNF-a, IL-6 or IL-1, the anti-TLR4 antibody did greatly inhibit the ability of LPS to stimulate cytokine production but not lipid S. The tetracyclines proved to be the most effective compounds, many were active at low concentrations and showed efficacy to inhibit both lipid S and LPS stimulated macrophages to produce NO.

Influence of iron deprivation and sub-inhibitory concentrations of antifungal antibiotics on surface antigens of candida albicans yeast cells

This study examined the effect of iron deprivation and sub-inhibitory concentrations of antifungal agents on yeast cell surface antigen recognition by antibodies from patients with Candida infections. Separation of cell wall surface proteins by sodium dodecyl-polyacrylamide gel electrophoresis (SDS-PAGE) and immunological detection by immunoblotting, revealed that antigenic profiles of yeasts were profoundly influenced by the growth environment. Cells grown under iron-depleted conditions expressed several iron-regulated proteins that were recognized by antibodies from patient sera. An attempt to characterize these proteins by lectin blotting with concanavalin A revealed that some could be glycoprotein in nature. Furthermore, these proteins which were located within cell walls and on yeast surfaces, were barely or not expressed in yeasts cultivated under iron-sufficient conditions. The magnitude and heterogeneity of human antibody responses to these iron-regulated proteins were dependent on the type of Candida infection, serum antibody class and yeast strain. Hydroxamate-type siderophores were also detected in supernatants of iron depleted yeast cultures. This evidence suggests that Candida albicans expresses iron-regulated proteins/glycoproteins in vitro which may play a role in siderophore-mediated iron uptake in Candida albicans. Sequential monitoring of IgG antibodies directed against yeast surface antigens during immunization of rabbits revealed that different antigens were recognized particularly during early and later stages of immunization in iron-depleted cells compared to iron-sufficient cells. In vitro and in vivo adherence studies demonstrated that growth phase, yeast strain and growth conditions affect adhesion mechanisms. In particular, growth under iron-depletion in the presence of sub-inhibitory concentrations of polyene and azole antifungals enhanced the hydrophobicity of C.albicans. Growth conditions also influenced MICs of antifungals, notably that of ketoconazole. Sub-inhibitory concentrations of amphotericin B and fluconazole had little effect on surface antigens, whereas nystatin induced profound changes in surface antigens of yeast cells. The effects of such drug concentrations on yeast cells coupled with host defence mechanisms may have a significant affect on the course of Candida infections.

The anti-inflammatory action of tetracyclines

For six decades tetracyclines have been successfully used for their broad spectrum antibiotic effects. However, non-antibiotic effects of tetracyclines have been reported. The anti-inflammatory effects of tetracycline drugs have been investigated in the context of a range of inflammatory diseases including sepsis and a number of neurodegenerative diseases. This thesis investigates the effects of a range of clinically important tetracyclines (oxytetracycline, doxycycline, minocycline and tigecycline) on the ability of the J774.2 cell line to produce nitric oxide when stimulated with the bacterial cell wall component, LPS. The proteome of J774.2 cells was analysed in response to LPS stimulation (1 µg/ml) with and without prior treatment with minocycline (50µg/ml), this allows the unbiased analysis of the cellular proteome in response to minocycline and LPS, protein spots of interest were excised and identified by nano-electrospray ionisation-linear ion trap mass spectroscopy. All of the tetracyclines that were investigated inhibited LPS-induced nitric oxide production in a dose dependent manner and this was due to the inhibition of inducible nitric oxide synthase expression. This is the first report to show that tigecycline inhibits inducible nitric oxide expression and nitric oxide production. Using two-dimensional gel electrophoresis and total protein staining eleven proteins were identified as being modulated by LPS. Of these eleven proteins; expression of some, but not all was modulated when the cells received a prior treatment with minocycline suggesting that minocycline does not completely block LPS-induced macrophage activation but probably specifically acts on particular inflammatory signaling pathways in macrophages. Three protein spots with a similar molecular weight but different pI values identified in this proteomic study were identified as ATP synthase ß chain. These different protein spots probably correspond to different phosphorylation states of the protein, suggesting that minocycline affects the balance of protein kinase and protein phosphatase activity in the immune response.

Improved production of industrially relevant recombinant proteins: application of multi-factorial design of experiments and scalable process modelling

The work described in this thesis focuses on the use of a design-of-experiments approach in a multi-well mini-bioreactor to enable the rapid establishments of high yielding production phase conditions in yeast, which is an increasingly popular host system in both academic and industrial laboratories. Using green fluorescent protein secreted from the yeast, Pichia pastoris, a scalable predictive model of protein yield per cell was derived from 13 sets of conditions each with three factors (temperature, pH and dissolved oxygen) at 3 levels and was directly transferable to a 7 L bioreactor. This was in clear contrast to the situation in shake flasks, where the process parameters cannot be tightly controlled. By further optimisating both the accumulation of cell density in batch and improving the fed-batch induction regime, additional yield improvement was found to be additive to the per cell yield of the model. A separate study also demonstrated that improving biomass improved product yield in a second yeast species, Saccharomyces cerevisiae. Investigations of cell wall hydrophobicity in high cell density P. pastoris cultures indicated that cell wall hydrophobin (protein) compositional changes with growth phase becoming more hydrophobic in log growth than in lag or stationary phases. This is possibly due to an increased occurrence of proteins associated with cell division. Finally, the modelling approach was validated in mammalian cells, showing its flexibility and robustness. In summary, the strategy presented in this thesis has the benefit of reducing process development time in recombinant protein production, directly from bench to bioreactor.

Isolation, characterisation and application of novel microbial protein cross-linking enzymes

Microbial transglutaminase is favoured for use in industry over the mammalian isoform, and hence has been utilized, to great effect, as an applied biocatalyst in many industrial areas including the food and textiles industries. There are currently only a limited number of microbial TGase sources known. A number of organisms have been screened for transglutaminase activity using biochemical assays directed towards TGase catalyzed reactions (amine incorporation and peptide cross-linking assay). Of those organisms screened, TGase was identified in a number of isolates including members of the Bacillus and Streptomyces families. In addition, a protein capable of performing a TGase-like reaction was identified in the organism Pseudomonas putida that was deemed immunologically distinct from previously described TGase isoforms, though further work would be required to purify the protein responsible. The genuses Streptoverticillium and Streptomyces are known to be closely related. A number of micro-organisms relating to Streptomyces mobaraensis (formerly Streptoverticillium mobaraensis) have been identified as harboring a TGase enzyme. The exact biological role of Streptomyces TGase is not well understood, though from work undertaken here it would appear to be involved in cell wall growth. Comparison of the purified Streptomyces TGase proteins showed them to exhibit marginally different characteristics in relation to enzymatic activity and pH dependency upon comparison with Streptomyces mobaraensis TGase. In addition, TGase was identified in the organism Saccharomonospora viridis that was found to be genetically identical to that from S. mobaraensis raising questions about the enzymes dissemination in nature. TGase from S. baldaccii was found to be most diverse with respect to enzymatic characteristics whilst still retaining comparable E(y-glutamyl) lysine bond formation to S. mobaraensis TGase. As such S. baldaccii TGase was cloned into an expression vector enabling mass production of the enzyme thereby providing a viable alternative to S. mobaraensis TGase for many industrial processes.

Peptidoglycan derived from Staphylococcus epidermidis induces Connexin43 hemichannel activity with consequences on the innate immune response in endothelial cells

Gram-positive bacterial cell wall components including PGN (peptidoglycan) elicit a potent pro-inflammatory response in diverse cell types, including endothelial cells, by activating TLR2 (Toll-like receptor 2) signalling. The functional integrity of the endothelium is under the influence of a network of gap junction intercellular communication channels composed of Cxs (connexins) that also form hemichannels, signalling conduits that are implicated in ATP release and purinergic signalling. PGN modulates Cx expression in a variety of cell types, yet effects in endothelial cells remain unresolved. Using the endothelial cell line b.End5, a 6 h challenge with PGN induced IL-6 (interleukin 6), TLR2 and Cx43 mRNA expression that was associated with enhanced Cx43 protein expression and gap junction coupling. Cx43 hemichannel activity, measured by ATP release from the cells, was induced following 15 min of exposure to PGN. Inhibition of hemichannel activity with carbenoxolone or apyrase prevented induction of IL-6 and TLR2 mRNA expression by PGN, but had no effect on Cx43 mRNA expression levels. In contrast, knockdown of TLR2 expression had no effect on PGN-induced hemichannel activity, but reduced the level of TLR2 and Cx43 mRNA expression following 6 h of PGN challenge. PGN also acutely induced hemichannel activity in HeLa cells transfected to express Cx43, but had no effect in Cx43-deficient HeLa OHIO cells. All ATP responses were blocked with Cx-specific channel blockers. We conclude that acute Cx43 hemichannel signalling plays a role in the initiation of early innate immune responses in the endothelium.

Phosphorus catalysis in the pyrolysis behaviour of biomass

Phosphorus is a key plant nutrient and as such, is incorporated into growing biomass in small amounts. This paper examines the influence of phosphorus, present in either acid (HPO) or salt ((NH)PO) form, on the pyrolysis behaviour of both Miscanthus × giganteus, and its cell wall components, cellulose, hemicellulose (xylan) and lignin (Organosolv). Pyrolysis-gas chromatography-mass spectrometry (PY-GC-MS) is used to examine the pyrolysis products during thermal degradation, and thermogravimetric analysis (TGA) is used to examine the distribution of char and volatiles. Phosphorus salts are seen to catalyse the pyrolysis and modify the yields of products, resulting in a large increase in char yield for all samples, but particularly for cellulose and Miscanthus. The thermal degradation processes of cellulose, xylan and Miscanthus samples occur in one step and the main pyrolysis step is shifted to lower temperature in the presence of phosphorus. A small impact of phosphorus was observed in the case of lignin char yields and the types of pyrolysis decomposition products produced. Levoglucosan is a major component produced in fast pyrolysis of cellulose. Furfural and levoglucosenone become more dominant products upon P-impregnation pointing to new rearrangement and dehydration routes. The P-catalysed xylan decomposition route leads to a much simpler mixture of products, which are dominated by furfural, 3-methyl-2-cyclopenten-1-one and one other unconfirmed product, possibly 3,4-dihydro-2-methoxy-2H-pyran or 4-hydroxy-5,6-dihydro-(2H)-pyran-2-one. Phosphorus-catalysed lignin decomposition also leads to a modified mixture of tar components and desaspidinol as well as other higher molecular weight component become more dominant relative to the methoxyphenyl phenols, dimethoxy phenols and triethoxy benzene. Comparison of the results for Miscanthus lead to the conclusion that the understanding of the fast pyrolysis of biomass can, for the most part, be gained through the study of the individual cell wall components, provided consideration is given to the presence of catalytic components such as phosphorus.

Intermediate pyrolysis and product identification by TGA and Py-GC/MS of green microalgae and their extracted protein and lipid components

The thermo-chemical conversion of green microalgae Chlamydomonas reinhardtii wild type (CCAP 11/32C), its cell wall deficient mutant C. reinhardtii CW15 (CCAP 11/32CW15) and Chlorella vulgaris (CCAP 211/11B) as well as their proteins and lipids was studied under conditions of intermediate pyrolysis. The microalgae were characterised for ultimate and gross chemical composition, lipid composition and extracted products were analysed by Thermogravimetric analysis (TG/DTG) and Pyrolysis-gaschromatography/mass-spectrometry (Py-GC/MS). Proteins accounted for almost 50% and lipids 16-22 % of dry weight of cells with little difference in the lipid compositions between the C. reinhardtii wild type and the cell wall mutant. During TGA analysis, each biomass exhibited three stages of decomposition, namely dehydration, devolatilization and decomposition of carbonaceous solids. Py-GC/MS analysis revealed significant protein derived compounds from all algae including toluene, phenol, 4-methylphenol, 1H-indole, 1H-indole-3methyl. Lipid pyrolysis products derived from C. reinhardtii wild type and C. reinhardtii CW15 were almost identical and reflected the close similarity of the fatty acid profiles of both strains. Major products identified were phytol and phytol derivatives formed from the terpenoid chain of chlorophyll, benzoic acid alkyl ester derivative, benzenedicarboxylic acid alkyl ester derivative and squalene. In addition, octadecanoic acid octyl ester, hexadecanoic acid methyl ester and hydrocarbons including heptadecane, 1-nonadecene and heneicosane were detected from C. vulgaris pyrolysed lipids. These results contrast sharply with the types of pyrolytic products obtained from terrestrial lignocellulosic feedstocks and reveal that intermediate pyrolysis of algal biomass generates a range of useful products with wide ranging applications including bio fuels.

Potassium catalysis in the pyrolysis behaviour of short rotation willow coppice

Short rotation willow coppice (SRC) and a synthetic biomass, a mixture of the basic biomass components (cellulose, hemicellulose and lignin), have been investigated for the influence of potassium on their pyrolysis behaviours. The willow sample was pre-treated to remove salts and metals by hydrochloric acid, and this demineralised sample was impregnated with potassium. The same type of pre-treatment was applied to components of the synthetic biomass. Characterisation was performed using thermogravimetric analysis with measurement of products by means of Fourier transform infrared spectroscopy (TGA-FTIR) and pyrolysis-gas chromatography-mass spectrometry (PY-GC-MS). A comparison of product distributions and kinetics are reported. While the general features of decomposition of SRC are described well by an additive behaviour of the individual components, there are some differences in the magnitude of the influence of potassium, and on the products produced. For both SRC and the synthetic biomass, TGA traces indicate catalytic promotion of both of the two-stages of biomass decomposition, and potassium can lower the average apparent first-order activation energy for pyrolysis by up to 50 kJ/mol. For both SRC and synthetic biomass the yields and distribution of pyrolysis products have been influenced by the presence of the catalyst. Potassium catalysed pyrolysis increases the char yields markedly and this is more pronounced for synthetic biomass than SRC. Gas evolution profiles during pyrolysis show the same general features for both SRC and synthetic biomass. Relative methane yields increase during the char formation stage of pyrolysis of the potassium doped samples. The evolution profiles of acetic acid and formaldehyde change, and these products are seen in lower relative amounts for both the demineralised samples. A greater variation in pyrolysis products is observed from the treated SRC samples compared to the different synthetic biomass samples. Furthermore, substituted phenols from lignin pyrolysis are more dominant in the pyrolysis profiles of the synthetic biomass than of the SRC, implying that the extracted lignins used in the synthetic biomass yield a greater fraction of monomeric type species than the lignocellulosic cell wall material of SRC. For both types of samples, PY-GS-MS analyses show that potassium has a significant influence on cellulose decomposition markers, not just on the formation of levoglucosan, but also other species from the non-catalysed mechanism, such as 3,4-dihydroxy-3-cyclobutene-1,2-dione. © 2007 Elsevier Ltd. All rights reserved.

Synthesis and screening of potential antimicrobial compounds

Tuberculosis (TB), an infection caused by human pathogen Mycobacterium tuberculosis, continues to kill millions each year and is as prevalent as it was in the pre-antimicrobial era. With the emergence of continuously-evolving multi-drug resistant strains (MDR) and the implications of the HIV epidemic, it is crucial that new drugs with better efficacy and affordable cost are developed to treat TB. With this in mind, the first part of this thesis discusses the synthesis of libraries of derivatives of pyridine carboxamidrazones, along with cyclised (1,2,4-triazole and 1,2,4-oxadiazole) and fluorinated analogues. Microbiological screening against M. tuberculosis was carried out at the TAACF, NIAID and IDRI (USA). This confirmed the earlier findings that 2-pyridyl-substituted carboxamidrazones were more active than the 4-pyridyl-substituted carboxamidrazones. Another important observation was that upon cyclisation of these carboxamidrazones, a small number of the triazoles retained their activity while in most of the remaining compounds the activity was diminished. This might be attributed to the significant increase in logP value caused by cyclisation of these linear carboxamidrazones, resulting in high lipophilicity and decreased permeability. Another reason might be that the rigidity conferred upon the compound due to cyclisation, results in failure of the compound to fit into the active site of the putative target enzyme. In order to investigate the potential change to the compounds’ metabolism in the organism and/or host, the most active compounds were selected and a fluorine atom was introduced in the pyridine ring. The microbiological results shows a drastic improvement in the activity of the fluorinated carboxamidrazone amides as compared to their non fluorinated counterpart. This improvement in the activity could possibly be the result of the increased cell permeability caused by the fluorine. In a subsidiary strand, a selection of long-chain , -unsaturated carboxylic esters, -keto, -hydroxy carboxylic esters and -keto, -hydroxy carboxylic esters, structurally similar to mycolic acids, were synthesised. The microbiological data revealed that one of the open chain compound was active against the Mycobacterium tuberculosis H37Rv strain and some resistant isolates. The possible compound activity could be its potential to disrupt mycobacterial cell wall synthesis by interfering with the FAS-II pathway.