Biotransformations of water insoluble substrates in aqueous, two-phase and encapsulated systems /

The biotransformation of water insoluble substrates by mammalian and bacterial cells has been problematic, since these whole cell reactions are primarily performed in an aqueous environment The implementation of a twophase or encapsulated system has the advantages of providing a low water system along with the physiological environment the cells require to sustain themselves. Encapsulation of mammalian cells by formation of polyamide capsules via interfacial polymerization illustrated that the cells could not survive this type of encapsulation process. Biotransformation of the steroid spironolactone [3] by human kidney carcinoma cells was performed in a substrate-encapsulated system, yielding canrenone [4] in 70% yield. Encapsulation of nitrile-metabolizing Rhodococcus rhodochrous cells using a polyamide membrane yielded leaky capsules, but biotransformation of 2-(4- chlorophenyl)-3-methylbutyronitrile (CPIN) [6] in a free cell system yielded CPIN amide [7] in 40% yield and 94% ee. A two-phase biotransformation of CPIN consisting of a 5:1 ratio of tris buffer, pH 7.2 to octane respectively, gave CPIN acid [8] in 30% yield and 97% ee. It was concluded that Rhodococcus rhodochrous ATCC 17895 contained a nonselective nitrile hydratase and a highly selective amidase enzyme.

Order and membrane organization in chlorhexidine-lipid mixtures /

Formulations of a general bactericidal agent, chlorhexidine, mixed with a phospholipid at different concentrations are investigated using ^H NMR spectroscopy on a chain-deuterated lipid analog. Lipid-chlorhexidine formulation is known to release the drug into an aqueous medium slowly, maintaining a comparable concentration of the drug for up to four times longer than a direct aqueous solution. The NMR data does not support the proposed liposomal entrapment of chlorhexidine in lipid compartments. Complex thermal history of the lipid-chlorhexidine preparations is investigated in detail. In preparation for a counterpart measurement, using ^H NMR of deuterated chlorhexidine mixed with protonated lipid, the synthesis of a deuterated analog of chlorhexidine is performed.

Skeletal muscle fibre-type comparison of whole tissue and subcellular membrane phospholipids and fatty acids

Membranes are dynamic structures that affect cell structure and function. Compositional changes ofmembranes have been shown with the application of a perturbation; however these are limited to whole tissue analysis. The purpose of this thesis was to compare the phospholipid (PL) fatty acid (FA) composition of rat whole muscle (Wm) to 1) purified and non-purified subsarcolemmal (SS) mitochondria in soleus, plantaris, and red gastrocnemius, and 2) sarcolemma, transverse-tubules, SS and intermyofibrillar (IMF) mitochondria fix)m whole hindlimb. The major findings were that 1) contamination significantly altered the PL FA composition of the SS mitochondrial membrane fraction, 2) Wm and SS mitochondria compositions differed between muscle types, and 3) Wm did not accurately reflect the PL FA composition of any isolated subcellular membranes, with each being unique from each other. As such, the relevancy of the trends reported in the literature of the effects of perturbations on Wm may be limited.

Host-parasite relations in a mycoparasitic system : alterations in membrane permeability and lipid composition in Choanephora cucurbitarum infected by Piptocephalis virginiana /

examined in Choanephora cucurbita rum during the early stages of infection by Piptocephalis virginiana » There was a small but consistent increase in the leakage of electrolytes, amino acids and sugars as a result of infection. These low levels of differential leakage in infected tissues are explained on the basis of the nature of this obligate, biotrophic, mycoparasitic system. Quantitative analysis of the twenty six amino acids and amino compounds detected in the leacheates — showed similar profiles in infected and control host and no new species of amino acids or amino compounds were detected in either infected or control host leacheates. Comparatively high amounts of aspartic acid, glutamic acid and alanine were found in the leacheates of host and infected host . Analyses of the sugars comprising the leacheates of infected and control host showed the presence of eight sugars, among which glucose was found in significant amounts (50-53%) ' The nutritional implication of this preferential leakage is discussed. No significant difference was observed in the leacheates of infected host sugar profiles compared with that of the control host. Profiles of the internal pool sugars of infected and control host did not reflect that obtained from the leacheate data, perhaps owing to leakage of sugars in a selective manner . Membrane lipid analyses yielded higher levels of lipid in infected host compared with the control, both at the 24 h and 36 h analyses. In addition, preliminary investigations of phosphorous-32 incorporation and turnover in phospholipids showed higher levels of 32p incorporation and turnover in infected host compared with the control. No apparent difference was noted in the profiles of the neutral lipid classes and the polar lipid classes of the membrane lipids as determined by one and two dimensional thin-layer chromatography respectively. However, a small but consistently higher degree of unsaturation was detected in the fatty acids of infected tissue compared with the control. Also, '^''-^^''^^'-'-^'^^c acid, a polyunsaturated fatty acid previously reported to show a direct correlation during the early stages of infection and the degree of parasitism of P. virginiana on C. cucurbitarum , was found in higher amounts in infected host membrane lipids compared with that of the control host. The implications of these membrane lipid alterations are discussed with particular reference to the small but consistently higher leakage of electrolytes, amino acids and sugars observed during infection in this study.

A comparative study of in vitro chitin synthase activity in mucoraceous hosts of a mycoparasite

A comparative study of in vitro chitin synthase activity in mucoraceous hosts of a mycoparasite: Chitin synthase, the enzyme responsible for the synthesis of chitin in fungal cell wall was extracted from young hyphae of Choanephora cucurbitarum and Phascolomyces articulosus, susceptible and resistant hosts, respectively, to the mycoparasite, Piptocephalis virginiana. Crude enzyme was identified and characterized by measuring the incorporation of the substrate [14C]-UDP-N-acetylglucosamine, into chitin. Most activity occurred in mixed membrane fraction. Inhibition of activity with Polyoxin D and activation with proteases, N-acetyl-glucosamine and magnesium and other ions was observed. Properties of the crude enzyme preparation such as cofactor requirement, Vmax , apparent Km value for UDP-GlcNAc, inhibition by Polyoxin D, response to pH and to temperature, and stability at 4°C were determined. Enzyme activity from both fungi displayed basically the same features as the corresponding enzymes reported from other mucoraceous fungi. However, the two preparations from P. articulosus and C. cucurbitarum differed from each other in their expressed activity (i.e., the preparations from ~ articulosus exhibited higher latency and higher specific chitin synthase activity than the corresponding preparations from ~ cucurbitarum). Trypsin was effective in activation only over a narrow concentration range. Acid protease was the most effec.tive activator. En.dogenous protease estimation indicated higher protease activity in C. cucurbitarum than in P. articulosus. The suggestion is made that regulation of chitin synthase activities may be related to host resistance in the mycoparasitic system.

Characterization of the phosphorylation of thylakoid membrane proteins from cyanobacterium synechococcus sp. PCC 6301 in light state 1 and light state 2

The distribution of excitation energy between the two photosystems (PSII and PSI) of photosynthesis is regulated by the light state transition. Three models have been proposed for the mechanism of the state transition in phycobilisome (PBS) containing organisms, two involving protein phosphorylation. A procedure for the rapid isolation of thylakoid membranes and PBS fractions from the cyanobacterium Synechococcus m. PCC 6301 in light state 1 and light state 2 was developed. The phosphorylation of thylakoid and soluble proteins rapidly isolated from intact cells in state 1 and state 2 was investigated. 77 K fluorescence emission spectra revealed that rapidly isolated thylakoid membranes retained the excitation energy distribution characteristic of intact cells in state 1 and state 2. Phosphoproteins were identified by gel electrophoresis of both thylakoid membrane and phycobilisome fractions isolated from cells labelled with 32p orthophosphate. The results showed very close phosphoprotein patterns for either thylakoid membrane or PBS fractions in state 1 and state 2. These results do not support proposed models for the state transition which required phosphorylation of PBS or thylakoid membrane proteins.

Effects of large anphiphilic ligands upon the spectra and kinetics of cytochrome C oxidase

Cytoch ro me c oxidase (ferrocytochrome c : 02 oxidoreductase ; EC 1.9. 3.1) is the terminal enzyme in the mitochondrial electron transport chain, catalyzing the transfer of electrons from ferrocytochrome c to molecular oxygen. The effects of two large amphiphilic molecules .. valinomycin and dibucaine upon the spectra of the isolated enzyme and upon the activity of both isolated enzyme and enzyme in membrane systems are investigated by using spectrophotometric and oxygen electrode techniques. The results show that both valinomycin and dibucaine change the Soret region of the spectrum and cause a partial inhibition in a concentration range higher than that in which they act as ionophores. It is concluded that both valinomycin and dibucain~ binding induce a conformational change of the protein structure which modifies the spectrum of the a3 CUB centre and diminishes the rate of electron transfer between cytochrome a and the binuclear centre.

Heterogeneity of photosystem II as it occurs in domain specific regions of the thylakoid membrane of spinach (spinacia oleracia L.)

Thylakoid membrane fractions were prepared from specific regions of thylakoid membranes of spinach (Spinacia oleracea). These fractions, which include grana (83), stroma (T3), grana core (8S), margins (Ma) and purified stroma (Y100) were prepared using a non-detergent method including a mild sonication and aqueous two-phase partitioning. The significance of PSlla and PSII~ centres have been described extensively in the literature. Previous work has characterized two types of PSII centres which are proposed to exist in different regions of the thylakoid membrane. a-centres are suggested to aggregate in stacked regions of grana whereas ~-centres are located in unstacked regions of stroma lamellae. The goal of this study is to characterize photosystem II from the isolated membrane vesicles representing different regions of the higher plant thylakoid membrane. The low temperature absorption spectra have been deconvoluted via Gaussian decomposition to estimate the relative sub-components that contribute to each fractions signature absorption spectrum. The relative sizes of the functional PSII antenna and the fluorescence induction kinetics were measured and used to determine the relative contributions of PSlla and PSII~ to each fraction. Picosecond chlorophyll fluorescence decay kinetics were collected for each fraction to characterize and gain insight into excitation energy transfer and primary electron transport in PSlla and PSII~ centres. The results presented here clearly illustrate the widely held notions of PSII/PS·I and PSlIa/PSII~ spatial separation. This study suggests that chlorophyll fluorescence decay lifetimes of PSII~ centres are shorter than those of PSlIa centres and, at FM, the longer lived of the two PSII components renders a larger yield in PSlIa-rich fractions, but smaller in PSIlr3-rich fractions.

Thermoacclimatory variations in the activities of enzymes implicated in ion transport in the rainbow trout, salmo gairdneri

Two groups of rainbow trout were acclimated to 20 , 100 , and 18 o C. Plasma sodium, potassium, and chloride levels were determined for both. One group was employed in the estimation of branchial and renal (Na+-K+)-stimulated, (HC0 3-)-stimulated, and CMg++)-dependent ATPase activities, while the other was used in the measurement of carbonic anhydrase activity in the blood, gill and kidney. Assays were conducted using two incubation temperature schemes. One provided for incubation of all preparations at a common temperature of 2S oC, a value equivalent to the upper incipient lethal level for this species. In the other procedure the preparations were incubated at the appropriate acclimation temperature of the sampled fish. Trout were able to maintain plasma sodium and chloride levels essentially constant over the temperature range employed. The different incubation temperature protocols produced different levels of activity, and, in some cases, contrary trends with respect to acclimation temperature. This information was discussed in relation to previous work on gill and kidney. The standing-gradient flow hypothesis was discussed with reference to the structure of the chloride cell, known thermallyinduced changes in ion uptake, and the enzyme activities obtained in this study. Modifications of the model of gill lon uptake suggested by Maetz (1971) were proposed; high and low temperature models resulting. In short, ion transport at the gill at low temperatures appears to involve sodium and chloride 2 uptake by heteroionic exchange mechanisms working in association w.lth ca.rbonlc anhydrase. G.l ll ( Na + -K + ) -ATPase and erythrocyte carbonic anhydrase seem to provide the supplemental uptake required at higher temperatures. It appears that the kidney is prominent in ion transport at low temperatures while the gill is more important at high temperatures. 3 Linear regression analyses involving weight, plasma ion levels, and enzyme activities indicated several trends, the most significant being the interrelationship observed between plasma sodium and chloride. This, and other data obtained in the study was considered in light of the theory that a link exists between plasma sodium and chloride regulatory mechanisms.

Effects of large anphiphilic ligands upon the spectra and kinetics of cytochrome C oxidase

Cytoch ro me c oxidase (ferrocytochrome c : 02 oxidoreductase ; EC 1.9. 3.1) is the terminal enzyme in the mitochondrial electron transport chain, catalyzing the transfer of electrons from ferrocytochrome c to molecular oxygen. The effects of two large amphiphilic molecules - valinomycin and dibucaine upon the spectra of the isolated enzyme and upon the activity of both isolated enzyme and enzyme in membrane systems are investigated by using spectrophotometric and oxygen electrode techniques. The results show that both valinomycin and dibucaine change the Soret region of the speetrum and cause a partial inhibition in a concentration range higher than that in which they act as ionophores. It is concluded that both valinomycin and dibucaine binding induce a conformational change of the protein structure which modifies the spectrum of the a3 CUB centre and diminishes the rate of electron transfer between cytochrome a and the binuclear centre.

X-ray diffraction of a gram-negative bacterial membrane mimetic

This thesis applies x-ray diffraction to measure he membrane structure of lipopolysaccharides and to develop a better model of a LPS bacterial melilbrane that can be used for biophysical research on antibiotics that attack cell membranes. \iVe ha'e Inodified the Physics department x-ray machine for use 3.'3 a thin film diffractometer, and have lesigned a new temperature and relative humidity controlled sample cell.\Ve tested the sample eel: by measuring the one-dimensional electron density profiles of bilayers of pope with 0%, 1%, 1G :VcJ, and 100% by weight lipo-polysaccharide from Pse'udo'lTwna aeTuginosa. Background VVe now know that traditional p,ntibiotics ,I,re losing their effectiveness against ever-evolving bacteria. This is because traditional antibiotic: work against specific targets within the bacterial cell, and with genetic mutations over time, themtibiotic no longer works. One possible solution are antimicrobial peptides. These are short proteins that are part of the immune systems of many animals, and some of them attack bacteria directly at the membrane of the cell, causing the bacterium to rupture and die. Since the membranes of most bacteria share common structural features, and these featuret, are unlikely to evolve very much, these peptides should effectively kill many types of bacteria wi Lhout much evolved resistance. But why do these peptides kill bacterial cel: '3 , but not the cells of the host animal? For gramnegative bacteria, the most likely reason is that t Ileir outer membrane is made of lipopolysaccharides (LPS), which is very different from an animal :;ell membrane. Up to now, what we knovv about how these peptides work was likely done with r !10spholipid models of animal cell membranes, and not with the more complex lipopolysa,echaricies, If we want to make better pepticies, ones that we can use to fight all types of infection, we need a more accurate molecular picture of how they \vork. This will hopefully be one step forward to the ( esign of better treatments for bacterial infections.

Cell wall degrading enzymes and interaction between Trichoderma Aggressivum and Agaricus Bisporus

Agaricus bisporus is the most commonly cultivated mushroom in North America and has a great economic value. Green mould is a serious disease of A. bisporus and causes major reductions in mushroom crop production. The causative agent of green mould disease in North America was identified as Trichoderma aggressivum f. aggressivum. Variations in the disease resistance have been shown in the different commercial mushroom strains. The purpose of this study is to continue investigations of the interactions between T. aggressivum and A. bisporus during the development of green mould disease. The main focus of the research was to study the roles of cell wall degrading enzymes in green mould disease resistance and pathogenesis. First, we tried to isolate and sequence the N-acetylglucosaminidase from A. bisporus to understand the defensive mechanism of mushroom against the disease. However, the lack of genomic and proteomic information of A. bisporus limited our efforts. Next, T. aggressivum cell wall degrading enzymes that are thought to attack Agaricus and mediate the disease development were examined. The three cell wall degrading enzymes genes, encoding endochitinase (ech42), glucanase (fJ-1,3 glucanase) and protease (prb 1), were isolated and sequenced from T. aggressivum f. aggressivum. The sequence data showed significant homology with the corresponding genes from other fungi including Trichoderma species. The transcription levels of the three T. aggressivum cell wall degrading enzymes were studied during the in vitro co-cultivation with A. bisporus using R T -qPCR. The transcription levels of the three genes were significantly upregulated compared to the solitary culture levels but were upregulated to a lesser extent in co-cultivation with a resistant strain of A. bisporus than with a sensitive strain. An Agrobacterium tumefaciens transformation system was developed for T. aggressivum and was used to transform three silencing plasmids to construct three new T. aggressivum phenotypes, each with a silenced cell wall degrading enzyme. The silencing efficiency was determined by RT-qPCR during the individual in vitro cocultivation of each of the new phenotypes with A. bisporus. The results showed that the expression of the three enzymes was significantly decreased during the in vitro cocultivation when compared with the wild type. The phenotypes were co-cultivated with A. bisporus on compost with monitoring the green mould disease progression. The data indicated that prbi and ech42 genes is more important in disease progression than the p- 1,3 glucanase gene. Finally, the present study emphasises the role of the three cell wall degrading enzymes in green mould disease infection and may provide a promising tool for disease management.

Intracellular antioxidant and DNA repair enzymes as correlates of stress resistance and longevity in vertebrates

In animals, both stress resistance and longevity appear to be influenced by the insulin/insulin-like growth factor-l signaling (lIS) pathway, the basic organization of which is highly conserved from invertebrates to vertebrates. Reduced lIS or genetic disruption of the lIS pathway leads to the activation of forkhead box transcription factors, which is thought to upregulate the expression of genes involved in enhancing stress resistance, including perhaps key antioxidant enzymes as well as DNA repair enzymes. Enhanced antioxidant and DNA repair capacities may underlie the enhanced cellular stress resistance observed in long-lived animals, however little data is available that directly supports this idea. I used three. experimental approaches to test the association of intracellular antioxidant and DNA base excision repair (BER) capacities with stress resistance and longevity: (1) a comparison of multiple vertebrate endotherm species of varying body masses and longevities; (2) a comparison of long-lived Snell dwarf mice and their normallittermates; and (3) a comparison of hypometabolic animals undergoing hibernation or estivation with their active counterparts. The activities of the five major intracellular antioxidant enzymes as well as the two rate-limiting enzymes in the BER pathway, apurininc/apyrimidinic (AP) endonuclease and polymerase ~, were measured. These measurements were performed in one or more of the following: (1) cultured dermal fibroblasts; (2) brain tissue; (3) heart tissue; (4) liver tissue. My results indicate that antioxidant enzymes are not universally upregulated in association with enhanced stress resistance and longevity. I also did not find that BER enzyme activity was positively correlated with longevity, in an inter-species context, though there was evidence for enhanced BER in long-lived Snell dwarf mice. Thus, while there were instances in which enhanced antioxidant and BER enzyme activities were associated with increased stress resistance and/or longevity, this was not universally the case, indicating that other mechanisms must be involved. These results suggest the need to re-examine existing 'oxidative stress' hypotheses of longevity and probe further into the molecular physiology of longevity to discover its mechanistic basis.