Inhibition of secretary activity in cells isolated from the rat stomach

The overall aim of this study was to further understanding of themechanisms by which inhibitors of secretory activity mediate their action inisolated stomach cells. One objective was to determine whether a G-proteinsensitive to inactivation by pertussis toxin was involved in the action of thefollowing inhibitors of histamine-stimulated acid secretion: prostaglandin E2(PGE2), somatostatin, epidermal growth factor (EGF) and 12-0-tetradecanoylphorbol 13-acetate (TPA), an activator of protein kinase C.The site and mechanism by which EGF inhibited acid secretion and itseffects on pepsinogen secretion were also of interest. Further objectiveswere to determine whether TPA could induce down-regulation of proteinkinase C in parietal cells and to examine the inhibitory action of cyclic GMPon acid secretion. Acid secretion was estimated by the accumulation of theweak base aminopyrine in parietal cells. Experiments in which cells were preincubated with pertussis toxinindicated that PGE2, somatostatin and EGF mediated their inhibitory actionagainst histamine-stimulation via an inhibitory G-protein of the "Gi·like"family. Stimulation of PGE2 production by EGF also involved a pertussistoxin-sensitive G-protein. EGF inhibited acid secretion stimulated byforskolin, but only in the absence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). This action of EGF was sensitive toinactivation by pertussis toxin. It is suggested that the effect of EGF was dueto an increase in low Km cyclic AMP phosphodiesterase activity, rather thanan effect on the histamine (H2) receptor. EGF did not inhibit pepsinogensecretion. TPA exerted only a small part of its inhibitory action by a mechanismsensitive to pertussis toxin. TPA was unable to induce detectable down-regulationof protein kinase C. Acid secretion stimulated by near-maximallyeffective concentrations of h1stamme plus IBMX, dibutyryl cyclic AMP(dbcAMP) and K+ was inhibited by dibutyryl cyclic GMP (dbcGMP).

An insight into the activation mechanism of the calcatonin-gene-related peptide receptor

The calcitonin-gene- related peptide (CGRP) receptor is unique among G-protein coupled receptors (GPCRs) as it consists of at least three proteins: calcitonin receptor like receptor (CLR), receptor activity modifying protein (RAMP)1 and receptor component protein (RCP). An endogenous agonist for this curious receptor is aCGRP, which is a sensory nerve-derived peptide made up of 37 amino acids. aCGRP acts as a potent vasodilator having pronounced effects on arterioles and capillaries. Understanding the pharmacodynamics of the CGRP receptor may have pharmaceutical benefit as the receptor has been associated with the onset of migraines and implicated in Raynauds syndrome. The primary aim of this thesis was to identify functionally important residues in the extracellular face of the CGRP receptor. Three areas of interest were selected including the extreme N-terminus of the CLR, extracellular loop 1 (ECL1) of the CLR and its associated transmembrane (TM) regions, and finally extracellular loop 3 (ECL3) of the CLR and its juxtamembrane regions. A site-directed mutagenesis (SDM) strategy was used to investigate these regions, primarily substituting the innate residues of CLR with alanine and assessing the mutation on multiple criteria including a functional cAMP assay, cell-surface expression, total expression, agonist-mediated internalisation and aCGRP binding. The results are interpreted and discussed taking into consideration contemporary concepts surrounding Secretin-like GPCRs. Moreover, the thesis also contains details of RAMP purification. Overall the thesis provides novel data that furthers insight into the complex phenomenon of CGRP receptor activation. Site-directed mutants have been identified that affect aCGRP binding, receptor signal transduction, the CLR/RAMP1 interface and the integrity of the protein complex structure.

Mechanism of uptake of the catecholic (7)-α-formamido cephalosporin BRL 41897A by klebsiella pneumoniae

The catecholic cephalosporin BRL 41897 A is resistant to β-lactamases and is taken up by bacteria via the iron transport system. The uptake of this antibiotic in E.coli uses the Fiu and Cir outer membrane proteins, whereas in P. aerugtnosa it enters via the pyochelin transport system. In this thesis mutants of K. pneumoniae resistant to BRL 41897A were isolated using TnphoA mutagenesis and used to study the mechanism of uptake of BRL 41897A by K. pneumoniae. The activity of BRL 41897A towards the parent strain (M10) was increased in iron depleted media, whereas no significant differences in the resistant (KSL) mutants were observed. Three mutants (KSL19, KSL38and KSL59) produced decreased amounts of certain iron-regulated outer membrane proteins. The uptake of 55Fe-BRL 41897A by M10 in iron-deficient medium was higher than in iron-rich medium. This result indicated the involvement of an iron transport system in the uptake of BRL 41897A by K. pneumoniae. Uptake by the KSL mutants in iron-deficient culture was higher than that by M10. This result, supported by analysis of outer membrane and periplasmic proteins of the KSL mutants, indicates that loss of one outer membrane protein can be compensated by over expression of other outer membrane and/or periplasmic proteins. However, the increased uptake of BRL 41897A by the KSL mutants did not reflect increased activity towards these strains, indicating that there are defects in the transport of BRL 41897A resulting in failure to reach the penicillin binding protein target sites in the cytoplasmic membrane. Southern blotting of chromosomal digests and sequencing in one mutant (KSL19) showed that only one copy of TnphoA was inserted into its chromosome. A putative TnphoA inserted gene in KSL19, designated kslA, carrying a signal sequence was identified. Transformation of a fragment containing the kslA gene into KSL19 cells restored the sensitivity to BRL 41897A to that of the parent strain. Data base peptide sequence searches revealed that the kslA gene in the KSL19 has some amino acid homology with the E. coli ExbD protein, which is involved in stabilisation of the TonB protein. 

Modulation of SERCA in the chronic phase of adjuvant arthritis as a possible adaptation mechanism of redox imbalance

Adjuvant arthritis (AA) is a condition that involves systemic oxidative stress. Unexpectedly, it was found that sarcoplasmic reticulum Ca2 +-ATPase (SERCA) activity was elevated in muscles of rats with AA compared to controls, suggesting possible conformational changes in the enzyme. There was no alteration in the nucleotide binding site but rather in the transmembrane domain according to the tryptophan polar/non-polar fluorescence ratio. Higher relative expression of SERCA, higher content of nitrotyrosine but no increase in phospholipid oxidation in AA SR was found. In vitro treatments of SR with HOCl showed that in AA animals SERCA activity was more susceptible to oxidative stress, but SR phospholipids were more resistant and SERCA could also be activated by phosphatidic acid. It was concluded that increased SERCA activity in AA was due to increased levels of SERCA protein and structural changes to the protein, probably induced by direct and specific oxidation involving reactive nitrogen species.

Mechanism of serca modulation from rats with adjuvant arthritis

We studied the structural and functional alterations of SERCA in rats suffering from adjuvant arthritis (AA). AA was induced by intradermal administration of Mycobacterium butyricum (MB) to the base of the tail of Lewis rats. Injury of SERCA from skeletal muscles of AA rats was analyzed on days 7, 14, 21 and 28 after MB injection. Neither fragmentation, aggregation of SERCA protein, alterations in SH groups, nor oxidation of phosphatidylcholines and phosphatidylethanolamines in SR vesicles were observed in animals with AA. The only ROS/RNS modification was increased formation of nitrotyrosine. The activity of SERCA from AA animals decreased on day 21 after MB injection and was associated with a significant increase of protein carbonyls in sarcoplasmic reticulum (SR). In contrast, on day 28 an increase of SERCA activity was observed and protein carbonyl level reversed to control level. Concerning kinetic parameters, maximum reaction velocity (Vmax) decrease and increase was observed with respect to both substrates (Ca, ATP) on days 21 and 28, respectively, suggesting possible conformational changes of the enzyme. These changes were not associated with alterations in nucleotide binding site situated in cytosol, but rather with tryptophan fluorescence intensity ratio (cytosol/membrane) related to the transmembrane domain of SERCA. Elevated SERCA activity on day 28 was caused by its higher expression. Acidic phospholipids (PA), probably present in SR of AA rats, may contribute to the elevation of Ca-ATPase activity, as PA administration in vitro increased this activity.

Mechanism of induction of muscle protein loss by hyperglycaemia

Treatment of murine myotubes with high glucose concentrations (10 and 25 mM) stimulated protein degradation through the ubiquitin–proteasome pathway, and also caused activation (autophosphorylation) of PKR (double-stranded-RNA-dependent protein kinase) and eIF2a (eukaryotic initiation factor 2a). Phosphorylation of PKR and eIF2a was also seen in the gastrocnemius muscle of diabetic ob/ob mice. High glucose levels also inhibited protein synthesis. The effect of glucose on protein synthesis and degradation was not seen in myotubes transfected with a catalytically inactive variant (PKR?6). High glucose also induced an increased activity of both caspase-3 and -8, which led to activation of PKR, since this was completely attenuated by the specific caspase inhibitors. Activation of PKR also led to activation of p38MAPK (mitogen activated protein kinase), leading to ROS (reactive oxygen species) formation, since this was attenuated by the specific p38MAPK inhibitor SB203580. ROS formation was important in protein degradation, since it was completely attenuated by the antioxidant butylated hydroxytoluene. These results suggest that high glucose induces muscle atrophy through the caspase-3/-8 induced activation of PKR, leading to phosphorylation of eIF2a and depression of protein synthesis, together with PKR-mediated ROS production, through p38MAPK and increased protein degradation.

Cytotoxic activity of Fagonia cretica against human breast cancer cells

In many parts of the world, plants are directly utilised for their medicinal properties. Traditional medicine from Pakistan, India and the Far East is well documented and its history is embedded in folklore. It has been documented that an aqueous extract of the desert shrub, Fagonia cretica, is a popular treatment for breast cancer in Pakistan. The administration of an aqueous extract of Fagonia cretica is reported effective at reducing tumour size and improving the quality of life of breast cancer patients, is well tolerated and does not exhibit adverse effects like vomiting, diarrhoea or alopecia which are common side effects of standard cytotoxic therapy. In the past, many pharmacologically active and chemotherapeutic compounds have been isolated from plants which subsequently have proven to be successful in clinical trials and been used as primary compounds in therapeutic regimes. Fagonia cretica has historical use as a treatment for breast cancer, yet there is little scientific evidence which shows chemotherapeutic potential towards breast tumours. Preparation and analysis of an aqueous extract of Fagonia cretica may reveal novel chemotherapeutic agents that can be used to effectively target cancer cells. An understanding of the mechanism of any activity may improve our understanding of cancer cell biology and reveal novel therapeutic targets. This thesis describes for the first time that an aqueous extract of Fagonia cretica shows potent in vitro cytotoxic activity towards breast cancer epithelial cell lines which was not seen towards normal mammary epithelial cells. Elucidation and characterisation of the cytotoxic mechanism was undertaken by analysing DNA damage, cell cycle status, apoptosis, metabolic state and expression of transcription factors and their targets. Finally, methods for the isolation and identification of active compound(s) were developed using various chromatographic techniques. An aqueous extract of Fagonia cretica was able to reduce cell viability significantly in two phenotypically different breast cancer cell lines (MCF-7 and MDA-MB-231). This activity was markedly reduced in normal mammary epithelial cells (HMEpC). Further investigation into the mode of action revealed that extract treatment induced cell cycle arrest and apoptosis in both MCF-7 and MDA-MB-231 cell lines. This coincided with the formation of DNA double stranded breaks and the DNA repair marker ?-H2AX. In MCF-7 cells, ATM/ATR activation resulted in increased p53 expression and of its transcriptional targets p21 and bax, suggesting a role for a p53-mediated response. Furthermore, inhibition of extract-induced p53 expression with siRNA reduced the cytotoxic effect against MCF-7 cells. Extract treatment was also associated with increased FOXO3a expression in MCF-7 and MDA-MB-231 cells. In the absence of functional p53, siRNA knockdown of extract-induced FOXO3a expression was completely abrogated, suggesting that FOXO3a plays a vital role in extract-induced cytotoxicity. Isolation and characterisation of the active compound(s) within the extract was attempted using liquid chromatography and mass spectrometry in conjunction with a cell viability assay. Multiple fractionations generated an active fraction that contained four major compounds as detected by mass spectrometry. However, none of these compounds were identified structurally or chemically due to constraints within the methodology.

Mechanism of activation of dsRNA-dependent protein kinase (PKR) in muscle atrophy

The role of Ca2+ in the activation of PKR (double-stranded-RNA-dependent protein kinase), which leads to skeletal muscle atrophy, has been investigated in murine myotubes using the cell-permeable Ca2+ chelator BAPTA/AM (1,2-bis (o-aminphenoxy) ethane-N,N,N',N'-tetraacetic acid tetra (acetoxymethyl) ester). BAPTA/AM effectively attenuated both the increase in total protein degradation, through the ubiquitin–proteasome pathway, and the depression of protein synthesis, induced by both proteolysis-inducing factor (PIF) and angiotensin II (Ang  II). Since both protein synthesis and degradation were attenuated this suggests the involvement of PKR. Indeed BAPTA/AM attenuated both the activation  (autophosphorylation) of PKR and the subsequent phosphorylation of eIF2a (eukaryotic initiation factor 2a) in the presence of PIF, suggesting the involvement of Ca2+ in this process. PIF also induced an increase in the activity of both caspases-3 and -8, which was attenuated by BAPTA/AM. The increase in caspase-3 and -8 activity was shown to be responsible for the activation of PKR, since the latter was completely attenuated by the specific caspase-3 and -8 inhibitors. These results suggest that Ca2+ is involved in the increase in protein degradation and decrease in protein synthesis by PIF and Ang II through activation of PKR by caspases-3 and -8.

Investigating the delivery of antimicrobial proteins and aminoglycoside antibiotics to the airways

Biopharmaceuticals are finding wide applications in the management of diverse disease conditions. Pulmonary delivery of proteins may constitute an effective and efficient non-invasive alternative to parenteral delivery, which is currently the main route of administration of biopharmaceutical drugs. A particular area, in which pulmonary delivery of peptides and proteins may find ready application, is in the local delivery of antimicrobial peptides and proteins to the airway, a measure that could potentially bring about improvements to currently available antipseudomonal therapies. This thesis has therefore sought to develop inhalable antimicrobial proteins in combination with antibiotics that have particularly good antimicrobial activity against Pseudomonas aeruginosa infections in the respiratory tract of people with cystic fibrosis (CF). Through process optimisation, a suitable spray drying method was developed and used for the preparation of active, inhalable dry powder formulations of the antimicrobial protein, lactoferrin, and aminoglycosides (tobramycin and gentamicin). The physicochemical properties, aerosolisation performance and the antibacterial properties of the various spray-dried formulations were assessed. In addition, a relevant in vitro cellular model was employed to investigate the potential cytotoxic and pro-inflammatory effects of the various formulations on four bronchial human epithelial cells together with their effectiveness at reducing bacterial colonies when administered on to biofilm co-cultured on the epithelial cells. It was found that following spray drying the particles obtained were mostly spherical, amorphous and possessed suitable aerosolisation characteristics. The various spray-dried antimicrobial proteins (lactoferrin or apo lactoferrin) and co-spray dried combinations of the proteins and aminoglycosides were found to exhibit bactericidal activity against planktonic and biofilms of P. aeruginosa. In general, the spray drying process was found not to significantly affect the antimicrobial activities of the protein. Treatment of the different bronchial epithelial cell lines with the antimicrobial formulations showed that the various formulations were non-toxic and that the co-spray dried combinations significantly reduced established P. aeruginosa biofilms on the four bronchial epithelial cells. Overall, the results from this thesis demonstrates that spray drying could potentially be employed to prepare inhalable antimicrobial agents comprised of proteins and antibiotics. These new combinations of proteins and aminoglycosides has promising applications in the management of P. aeruginosa in the airway of cystic fibrosis patients.

Mechanism of attenuation of angiotensin-II-induced protein degradation by insulin-like growth factor-I (IGF-I)

Insulin-like growth factor-I (IGF-I) has been shown to attenuate protein degradation in murine myotubes induced by angiotensin II through downregulation of the ubiquitin-proteasome pathway, although the mechanism is not known. Angiotensin II is known to upregulate this pathway through a cellular signalling mechanism involving release of arachidonic acid, activation of protein kinase Cα (PKCα), degradation of inhibitor-κB (I-κB) and nuclear migration of nuclear factor-κB (NF-κB), and all of these events were attenuated by IGF-I (13.2 nM). Induction of the ubiquitin-proteasome pathway has been linked to activation of the RNA-activated protein kinase (PKR), since an inhibitor of PKR attenuated proteasome expression and activity in response to angiotensin II and prevented the decrease in the myofibrillar protein myosin. Angiotensin II induced phosphorylation of PKR and of the eukaryotic initiation factor-2 (eIF2) on the α-subunit, and this was attenuated by IGF-I, by induction of the expression of protein phosphatase 1, which dephosphorylates PKR. Release of arachidonic acid and activation of PKCα by angiotensin II were attenuated by an inhibitor of PKR and IGF-I, and the effect was reversed by Salubrinal (15 μM), an inhibitor of eIF2α dephosphorylation, as was activation of PKCα. In addition myotubes transfected with a dominant-negative PKR (PKRΔ6) showed no release of arachidonate in response to Ang II, and no activation of PKCα. These results suggest that phosphorylation of PKR by angiotensin II was responsible for the activation of the PLA2/PKC pathway leading to activation of NF-κB and that IGF-I attenuates protein degradation due to an inhibitory effect on activation of PKR. © 2007 Elsevier Inc. All rights reserved.

Mechanism of induction of muscle protein degradation by angiotensin II

Angiotensin I and II have been shown to directly induce protein degradation in skeletal muscle through an increased activity and expression of the ubiquitin-proteasome proteolytic pathway. This investigation determines the role of the nuclear transcription factor nuclear factor-κB (NF-κB) in this process. Using murine myotubes as a surrogate model system both angiotensin I and II were found to induce activation of protein kinase C (PKC), with a parabolic dose-response curve similar to the induction of total protein degradation. Activation of PKC was required for the induction of proteasome expression, since calphostin C, a highly specific inhibitor of PKC, attenuated both the increase in total protein degradation and in proteasome expression and functional activity increased by angiotensin II. PKC is known to activate I-κB kinase (IKK), which is responsible for the phosphorylation and subsequent degradation of I-κB. Both angiotensin I and II induced an early decrease in cytoplasmic I-κB levels followed by nuclear accumulation of NF-κB. Using an NF-κB luciferase construct this was shown to increase transcriptional activation of NF-κB regulated genes. Maximal luciferase expression was seen at the same concentrations of angiotensin I/II as those inducing protein degradation. Total protein degradation induced by both angiotensin I and II was attenuated by resveratrol, which prevented nuclear accumulation of NF-κB, confirming that activation of NF-κB was responsible for the increased protein degradation. These results suggest that induction of proteasome expression by angiotensin I/II involves a signalling pathway involving PKC and NF-κB. © 2005 Elsevier Inc. All rights reserved.

The coupling mechanism of P-glycoprotein involves residue L339 in the sixth membrane spanning segment

The transmembrane (TM) domains in P-glycoprotein (P-gp) contain the drug binding sites and undergo conformational changes driven by nucleotide catalysis to effect translocation. However, our understanding of exactly which regions are involved in such events remains unclear. A site-directed labelling approach was used to attach thiol-reactive probes to cysteines introduced into transmembrane segment 6 (TM6) in order to perturb function and infer involvement of specific residues in drug binding and/or interdomain communication. Covalent attachment of coumarin-maleimide at residue 339C within TM6 resulted in impaired ATP hydrolysis by P-gp. The nature of the effect was to reduce the characteristic modulation of basal activity caused by transported substrates, modulators and the potent inhibitor XR9576. Photoaffinity labelling of P-gp with [(3)H]-azidopine indicated that residue 339C does not alter drug binding per se. However, covalent modification of this residue appears to prevent conformational changes that lead to drug stimulation of ATP hydrolysis.

Mechanism of attenuation of protein loss in murine C2C12 myotubes by d-myo-inositol 1,2,6-triphosphate

d-Myo-inositol 1,2,6-triphosphate (alpha trinositol, AT) has been shown to attenuate muscle atrophy in a murine cachexia model through an increase in protein synthesis and a decrease in degradation. The mechanism of this effect has been investigated in murine myotubes using a range of catabolic stimuli, including proteolysis-inducing factor (PIF), angiotensin II (Ang II), lipopolysaccharide, and tumor necrosis factor-α/interferon-γ. At a concentration of 100 μM AT was found to attenuate both the induction of protein degradation and depression of protein synthesis in response to all stimuli. The effect on protein degradation was accompanied by attenuation of the increased expression and activity of the ubiquitin-proteasome pathway. This suggests that AT inhibits a signalling step common to all four agents. This target has been shown to be activation (autophosphorylation) of the dsRNA-dependent protein kinase (PKR) and the subsequent phosphorylation of eukaryotic initiation factor 2 on the α-subunit, together with downstream signalling pathways leading to protein degradation. AT also inhibited activation of caspase-3/-8, which is thought to lead to activation of PKR. The mechanism of this effect may be related to the ability of AT to chelate divalent metal ions, since the attenuation of the increased activity of the ubiquitin-proteasome pathway by PIF and Ang II, as well as the depression of protein synthesis by PIF, were reversed by increasing concentrations of Zn2+. The ability of AT to attenuate muscle atrophy by a range of stimuli suggests that it may be effective in several catabolic conditions. © 2009 Elsevier Inc. All rights reserved.

In vitro protective effect and antioxidant mechanism of Resveratrol induced by Dapsone Hydroxylamine in human cells

Dapsone (DDS) hydroxylamine metabolites cause oxidative stress- linked adverse effects in patients, such as methemoglobin formation and DNA damage. This study evaluated the ameliorating effect of the antioxidant resveratrol (RSV) on DDS hydroxylamine (DDSNHOH) mediated toxicity in vitro using human erythrocytes and lymphocytes. The antioxidant mechanism was also studied using in-silico methods. In addition, RSV provided intracellular protection by inhibiting DNA damage in human lymphocytes induced by DDS-NHOH. However, whilst pretreatment with RSV (10-1000 μM significantly attenuated DDS-NHOH-induced methemoglobinemia, but it was not only significantly less effective than methylene blue (MET), but also post-treatment with RSV did not reverse methemoglobin formation, contrarily to that observed with MET. DDS-NHOH inhibited catalase (CAT) activity and reactive oxygen species (ROS) generation, but did not alter superoxide dismutase (SOD) activity in erythrocytes. Pretreatment with RSV did not alter these antioxidant enzymes activities in erythrocytes treated with DDS-NHOH. Theoretical calculations using density functional theory methods showed that DDS-NHOH has a pro-oxidant effect, whereas RSV and MET have antioxidant effect on ROS. The effect on methemoglobinemia reversion for MET was significantly higher than that of RSV. These data suggest that the pretreatment with resveratrol may decrease heme-iron oxidation and DNA damage through reduction of ROS generated in cells during DDS therapy.

A cell-permeable tool for analysing APP intracellular domain function and manipulation of PIKfyve activity

The mechanisms for regulating PIKfyve complex activity are currently emerging. The PIKfyve complex, consisting of the phosphoinositide kinase PIKfyve (also known as FAB1), VAC14 and FIG4, is required for the production of phosphatidylinositol-3,5-bisphosphate (PI(3,5)P2). PIKfyve function is required for homeostasis of the endo/lysosomal system and is crucially implicated in neuronal function and integrity, as loss of function mutations in the PIKfyve complex lead to neurodegeneration in mouse models and human patients. Our recent work has shown that the intracellular domain of the Amyloid Precursor Protein (APP), a molecule central to the aetiology of Alzheimer's disease binds to VAC14 and enhances PIKfyve function. Here we utilise this recent advance to create an easy-to-use tool for increasing PIKfyve activity in cells. We fused APP's intracellular domain (AICD) to the HIV TAT domain, a cell permeable peptide allowing proteins to penetrate cells. The resultant TAT-AICD fusion protein is cell permeable and triggers an increase of PI(3,5)P2. Using the PI(3,5)P2 specific GFP-ML1Nx2 probe we show that cell-permeable AICD alters PI(3,5)P2 dynamics. TAT-AICD also provides partial protection from pharmacological inhibition of PIKfyve. All three lines of evidence show that the APP intracellular domain activates the PIKfyve complex in cells, a finding that is important for our understanding of the mechanism of neurodegeneration in Alzheimer's disease.