No description available.

Cardiac hypertrophy often develops to compensate for hemodynamic overload and therefore, in its early stages, hypertrophy is considered to be an adaptive response. Nonetheless, prolonged exposure to a hypertrophic stimulus is associated with heart failure. In the heart, the compartmentalisation of the cAMP/ PKA signalling pathway plays a critical role to achieve the specificity of response and maintains regular cardiac function. Alterations in this signalling pathway have been linked to the pathophysiology of cardiac hypertrophy. Phosphodiesterases (PDEs) provide the only means of hydrolysing cAMP and therefore are essential components in the spatial and temporal control of the cAMP response. By restricting the diffusion of cAMP, PDEs prevent unspecific activation of PKA and phosphorylation of downstream targets. PDEs are therefore able to regulate the kinetics of cAMP signalling dynamics. In this study, an in vitro model of chronic catecholamine-induced cardiac hypertrophy of adult rat ventricular myocytes (ARVM) was utilised. This model allowed the investigation of the function of PDEs in regulating compartmentalised cAMP signals in cardiac hypertrophy. Using FRET-based cAMP sensor Epac1_camps fused to the unique dimerisation/docking domain sequences that anchors PKA‐RI and PKA‐RII subunits to AKAPs, this study demonstrated that, similar to neonatal rat ventricular myocytes (NRVM), adult myocytes also display restricted cAMP diffusion. These cAMP microdomains are regulated by different families of PDEs. In particular, PDE2, PDE3 and PDE4 appear to control the pool of cAMP generated in the PKA-RI compartment, whereas only PDE2 and PDE4 were found to modulate cAMP in the PKA-RII compartment in ARVM. In the in vitro cardiac hypertrophy model, a reduction in cAMP generation was detected upon β-adrenergic stimulation and altered PDE activity was visualised using FRET-based imaging. This investigation showed that PDE2 activity is significantly increased in the PKA-RII compartment of hypertrophic cardiac myocytes, while an overall reduction in PDE3 activity was detected. Immunofluorescence experiments revealed altered PDE4 localisation in hypertrophic myocytes. Advances in cyclic nucleotide signalling research, in particular of the activity and regulation of PDEs, have shown that an interaction between the cAMP and cGMP signalling pathways exists. Integration between these two pathways is mediated by the modulation of cAMP‐degrading PDEs by cGMP. Allosteric binding of cGMP to the GAF domains of PDE2 enhances its activity, whereas cGMP reduces the activity of PDE3 by acting as a competitive inhibitor. PDE2 and PDE3 therefore may act as a connection between these two signalling cascades and it is possible to predict the existence of distinct signalling units in vivo in which cGMP, by acting on PDE2 or PDE3, can selectively modulate cAMP levels. Intracellular cGMP generated by stimulation of the particulate GC (pGC) by atrial natriuretic peptide (ANP) or stimulation of the soluble GC (sGC) by the NO donor SNAP is compartmentalised into discrete microdomains. Stimulation of the pGC had no effect on cAMP signalling in the PKA-RII compartment. Activation of sGC generated a pool of cGMP which lead to a reduction in cAMP response in the PKA-RII compartment upon β-AR stimulation. Both GCs generated cGMP in the PKA-RI compartment which lead to an increase in cAMP response. Further investigation revealed that cGMP is able to modulate cAMP signalling in the PKA-RI compartment by PDE3 and by PDE2 in the PKA-RII compartment. It was hypothesised that the observed differences in cAMP signalling and PDE contribution in hypertrophic myocytes in the PKA-RI and PKA-RII subcellular compartments may be due to variations in the pools of cGMP. Employing genetically encoded FRET-based biosensors for cGMP targeted to PKA‐RI and PKA‐RII compartments, basal levels of cGMP were found to be significantly increased in both compartments of hypertrophic myocytes which could explain the altered PDE2 and PDE3 activity in hypertrophic ARVM. Finally, this study shows that PDE2 activity is necessary to achieve full development of the catecholamine-induced hypertrophic response. Pharmacological inhibition of PDE2 with Bay 60-7550 prevents NE-induced cardiomyocyte hypertrophy. Overexpression of PDE2A2 was found to induce hypertrophic growth, even in the absence of increased adrenergic drive, thus indicating that PDE2 activity promotes development of cardiomyocytes hypertrophy. PDE2 localisation is vital for its regulation of hypertrophic growth. Displacement of endogenously active PDE2 from its specific intracellular localisation, using a catalytically inactive PDE2A2, was sufficient to counteract catecholamine-induced hypertrophic growth As PDE2 is a dual specific PDE, it was important to establish whether the anti-hypertrophic effects of PDE2 inhibition were mediated by activation of the cGMP/ PKG or cAMP/ PKA signalling pathways. The data presented here, show that the effects of pharmacological inhibition of PDE2 is PKA dependent. Together, these findings confirm the involvement of PDE2 in the progression of hypertrophy in cardiomyocytes and indentify PDE2, specifically coupled to the PKA-RII compartment, as a possible novel target for the development of therapeutic treatment for hypertrophy.

Thiol dependent reductase 1 (TDR1) of Leishmania has been implicated in both the activation of pentavalent antimonial drugs and in the generation of drug-thiol conjugates facilitating drug-resistance. Reverse genetic studies were carried out on TDR1 to elucidate the role of the enzyme and to assess its potential value as a drug target against Leishmania. In a similar study, O¬acetylserine (thiol) lyase (OAS-TL), a key enzyme for the de novo synthesis of cysteine in Leishmania, was investigated as a potential target for new antileishmanial drugs. TDR1 of Leishmania is a 49.9 kDa protein of which the physiological role remains unclear. The protein has shown both thiol transferase activity and dehydroascorbate reductase activity. Due to its ability to reduce pentavalent antimonials to the active trivalent form in vitro, TDR1 has been suggested as playing a vital role in antimonial resistance in Leishmania. This part of the study was undertaken to clarify the role TDR1 plays in the parasite by investigating the effects of deleting the gene. Attempts were made to generate Δtdr1 null mutants in Leishmania donovani, but these were unsuccessful despite the fact that Δtdr1 null mutants exist for L. major and L. infantum. The mutants of these latter lines were studied to discover more on the roles of the proteins. L. major and L. infantum Δtdr1 null mutant promastigotes grow normally and do not display any change in total intracellular levels of cysteine, glutathione and trypanothione. The L. major Δtdr1 null mutants were able to survive and proliferate in parasitophorous vacuoles of peritoneal macrophages in vitro, with significantly higher numbers of parasites per infected macrophage compared to L. major wild-type. This suggests that the loss of TDR1 is beneficial to L. major when establishing an infection in macrophages. However the loss of TDR1 also causes hypersensitivity to the antimonial drug sodium stibogluconate, under the conditions tested. The data generated in this study indicate that the physiological function of TDR1 does not lie in the activation of pentavalent antimonials as has been previously suggested. The sulfur-containing amino acid cysteine plays a vital role in the synthesis of low molecular weight thiols, e.g. glutathione and trypanothione, as well as redox active thiol-containing proteins. In addition, cysteine is important for the stabilisation of tertiary and quaternary protein conformation due to its ability to form inter- and intra-chain disulfide bonds with other cysteine residues. In mammals, cysteine can either be taken up from the environment, or synthesised via the reverse trans-sulfuration pathway, involving the action of the enzymes cystathionine β-synthase and cystathionine γ-lyase, to generate cysteine from the essential amino acid methionine. In contrast, Leishmania parasites can synthesise cysteine in two ways but appear unable to salvage it effectively. They contain the reverse trans-sulfuration pathway, similar to mammals, and additionally, can generate cysteine through the sulfhydrylation pathway from serine, coenzyme A and sulfide, by utilising the enzymes serine acetyltransferase and O-acetylserine (thiol) lyase (OAS-TL). The aim of this study was to assess the suitability of OAS-TL as a potential drug target against Leishmania. In this study, Δoas-tl null mutants were generated in L. donovani, thus negating the sulfhydrylation pathway. The Δoas-tl null mutant promastigotes displayed a slight growth defect as well as a severe morphological alterations directly affecting cell body and flagellum length. In addition, the Δoas-tl null mutants were unable to survive in the parasitophorous vacuoles of peritoneal macrophages in vitro, suggesting that the exogenous supply of a source of cysteine (such as methionine) was not sufficiently high to support parasite proliferation. The finding that addition of high methionine concentrations to the medium facilitates parasite survival supports this idea. The data show that either differentiation of promastigotes into amastigotes or proliferation of amastigotes is detrimentally affected by the deletion of OAS-TL. Lines re-expressing OAS-TL were also generated in the Δoas-tl null mutants and were found to complement the phenotypes of the Δoas-tl null mutants identified in this study. The inability of Leishmania Δoas-tl null mutants to survive within macrophages, together with the absence of OAS-TL in the mammalian host, make it a suitable candidate for the identification of new drug targets in the search for novel chemotherapeutic agents against leishmaniasis.

Abstract P.aeruginosa is an extracellular, Gram-negative opportunistic pathogen. One of the most important virulence factors during infection is the type III secretion system (T3SS). This system is found exclusively in Gram-negative bacteria and it forms a conduit between the bacteria and the host cell through which effector molecules can be translocated. These effectors alter the function of the host cell to promote survival of the bacterium. Infections are detected initially by the innate immune system via germ-line encoded receptors, pathogen recognition receptors (PRRs). These receptors recognise conserved microbial patterns, known as pathogen-associated molecular patterns and molecules which signal danger, danger-associated molecular patterns. PRRs are both membrane bound, such as Toll-like receptors (TLRs), and cytosolic, such as Nod-like receptors (NLRs). Some NLRs are involved in the formation of multimeric protein complexes, the Nod-signalosome and inflammasomes. These lead to the activation of NF-κB and the activation of caspase-1 and subsequent proteolytic processing of interleukin-1β (IL-1β) into its mature form. Both processes contribute to the inflammatory response following infection. In this study we sought to elucidate whether P.aeruginosa is able to trigger cytosolic PRRs and the mechanism of this activation. Initially we studied inflammasome activation by P.aeruginosa. We demonstrated that P.aeruginosa is able to activate the NLRC4/ASC-inflammasome complex. This was found to be dependent on a functional T3SS, but independent of any effectors passing through the system. The activation was discovered by detection of processed, and thus active caspase-1 fragments, as well as by secretion of mature IL-1β. The mechanism of the inflammasome activation was then investigated. We found that the NLRC4-dependent inflammasome activation is also dependent on extracellular potassium. An increase of extracellular potassium leads to a complete abrogation of inflammasome activation by P.aeruginosa and Salmonella. To further elucidate this finding, we investigated the leakiness of the pore formed by the T3SS in the host cell membrane. No flux of ions or small molecules could be detected in the host cell membrane following infection. However, host-membrane repair mechanisms were triggered, which could be detected by lysosomal-associated membrane protein (LAMP)-1-specific staining of the plasma membrane. We hypothesize a role for membrane potential in triggering of inflammasome activation by bacteria possessing a secretion system. Potassium-efflux has previously been identified as a activator of the NLRP3 inflammasome, but no changes in intracellular potassium could be found during this study. The activation of the NLRC4 inflammasome by the Pseudomonal strain PA103 was shown, in this study, to be independent of flagellin. Instead, the bacterial molecule responsible for inflammasome activation was shown to be pilin. Pilin is important for attachment to the host cell and the function of the T3SS. We showed that a strain lacking pilin were still able to translocate effectors through its T3SS. However, it was unable to activate the inflammasome complex. Transfection of purified pilin into cells was shown to trigger inflammasome activation. This was found to be dependent on caspase-1 but independent of NLRC4 and ASC, which is not in agreement with the results found for live bacteria. We hypothesised that the reason for this is the delivery method used, since a T3SS and infection delivers proteins and molecules differently compared to a transfection reagent. Finally, the role for Nod1 in infection by P.aeruginosa was explored. We could not identify Nod1-dependent NF-κB-activation using luciferase reporter gene assays. We therefore hypothesise that Nod1 does not have a role in the innate immune response to P.aeruginosa. In conclusion, we have identified NLRC4- and ASC-dependent inflammasome activation by P.aeruginosa. This activation was shown to be dependent on a functional T3SS and the surface protein pilin, as well as extracellular potassium. This describes a novel NLRC4-activation mechanism dependent on potassium and identifies pilin as a PRR-trigger for the first time.

AMP-activated protein kinase (AMPK) has been proposed to be a therapeutic target for patients with type 2 diabetes and the metabolic syndrome. In skeletal muscle AMPK stimulates glucose uptake and fatty acid oxidation, whereas in liver it inhibits fatty acid and cholesterol synthesis. The Rab GTPase activating proteins Akt substrate of 160 kDa (AS160) and tre-2/USP6, BUB2, cdc16 domain family member 1 (TBC1C1) have been identified as potential targets of both protein kinase B (PKB, also known as Akt) and AMPK which mediate glucose transporter 4 (GLUT4) translocation to the plasma membrane in response to insulin and 5-aminoimidazole-4-carboxamide riboside (AICAR) respectively in muscle. Previous work in our laboratory has demonstrated that AICAR modestly stimulates basal glucose transport, yet inhibits insulin-stimulated glucose transport in 3T3-L1 adipocytes, which is in contrast to the effect of AICAR in skeletal muscle. Currently the role of AMPK in adipocytes remains poorly characterised despite the importance of fat tissue in energy homeostasis. To address this, the molecular mechanism of AMPK activation by known stimuli, the acute effect of various AMPK activators on glucose transport, the effect of AMPK inhibition and knockdown on AICAR mediated inhibition of insulin-stimulated glucose transport and the effect of acute AICAR treatment on PKB substrate phosphorylation in 3T3-L1 adipocytes was investigated. In addition the effect of sustained AMPK activation on glucose transport and insulin signaling in 3T3-L1 adipocytes, and the effect of sustained AMPK activation on insulin signaling in human adipose tissue was also investigated. The AMPK activators; sorbitol, metformin, rosiglitazone, arsenite, azide, hydrogen peroxide and isoproterenol were all shown to stimulate AMPK activity in the presence of the Ca2+/Calmodulin dependent protein kinase kinase (CaMKK) inhibitor STO-609, suggesting that these activators activate AMPK via a CaMKK-independent pathway in 3T3-L1 adipocytes. However, A23187-stimulated AMPK activity was abrogated in the presence of STO-609. Isoproterenol, sodium azide and rosiglitazone, were all shown to cause an increase in the ADP/ATP ratio in 3T3-L1 adipocytes compared to control as assessed by high performance liquid chromatography, suggesting that they stimulate AMPK activity in an LKB1-dependent manner. These results suggest a possible role for CaMKK as an upstream AMPK kinase in 3T3-L1 adipocytes, in addition to LKB1. There may also exist other upstream AMPK kinases in 3T3-L1 adipocytes that are both nucleotide and calcium independent since sorbitol, metformin, arsenite, hydrogen peroxide and leptin were found to activate AMPK independently of CaMKK and also showed no significant effect on adenine nucleotide ratios. Sorbitol, rosiglitazone, AICAR, isoproterenol and A769662 all significantly inhibited insulin-stimulated glucose transport. Furthermore, in the presence of the AMPK inhibitor, Compound C, the inhibitory effect of AICAR on insulin-stimulated glucose transport was no longer apparent. However, AICAR still displayed a tendency to inhibit insulin-stimulated glucose transport in 3T3-L1 adipocytes infected with adenoviruses expressing a dominant negative AMPK mutant.The effect of AICAR on basal and insulin-stimulated AS160/TBC1D1 phosphorylation at phospho-Akt substrate (PAS) sites, was assessed. AICAR did not alter AS160/TBC1D1 phosphorylation compared to basal levels, nor perturb insulin-stimulated AS160/TBC1D1 phosphorylation at PAS sites. In addition, AICAR did not appear to alter the phosphorylation of any other proteins at PAS sites. Prolonged AMPK activation by AICAR in 3T3-L1 adipocytes also significantly inhibited insulin-stimulated glucose transport and was not associated with altered PKB protein expression or insulin-stimulated PKB Ser473 phosphorylation. In addition, chronic AMPK activation by metformin in adipose tissue of type 2 diabetic subjects was not associated with altered expression of three key insulin signalling molecules; PKB, the phosphoinositide 3-kinase (PI3K) p85 subunit and insulin receptor substrate 1 (IRS-1). Overall these results suggest a prominent role for LKB1 as an AMPK kinase and a potential role for CaMKK as an AMPK kinase in adipocytes. This study also suggests that both acute and prolonged AMPK activation in adipocytes inhibits insulin-stimulated glucose uptake, however the precise mechanism of inhibition has yet to be elucidated.

No description available.

No description available.

No description available.

Cancer is a leading cause of death worldwide as cancer cells have the ability to develop resistance to chemotherapeutic agents due to their high expressions of resistance gene. More research on alternative strategies to eradicate this type of malignant cells is highly desired by using natural products with fewer side effects. This study initially investigates the cytotoxicity of 20 novel semi-synthetic cardamonin derivatives and complexes in A549 lung and HK1 nasopharyngeal cancer cell lines. Structure activity relationship analysis revealed the factors affecting cytotoxicity were modification of hydroxyl group, presence of alkene group, addition of chemical groups, with the greatest cytotoxicity advantage being complexing with metal ions. Cardamonin-Cu(II) and cardamonin-Fe(II) complexes were determined as the two most cytotoxic compounds and were selected for further cytotoxic analysis in normal MRC5 lung and normal Hs68 foreskin cell lines. Results showed toxicity in MRC5 lung cells but was less toxic in Hs68 foreskin cells, suggesting some level of selectivity from cardamonin, cardamonin-Cu(II) and cardamonin-Fe(II) depending on the type of cells exposed to. Subsequently, cardamonin and its two complexes were tested in Jurkat T leukaemic cells and results showed highest susceptibility in this leukaemic cell line prompting further investigation on differential susceptibility of adherent and suspension cells. THP-1 monocytic leukaemia cell line cultured in both suspension and adherent phase demonstrated increased resistance in THP-1-derived macrophages in adherent phase compared to THP-1 monocytes in suspension form. Herein, we investigated the effects of interference from inhibitors of p38α and p38β MAP kinase and found THP-1-derived macrophages’ increased resistant towards cardamonin, cardamonin-Cu(II) and cardamonin-Fe(II) complexes were independent of p38α and p38β MAP kinase pathway. As Jurkat T cells exhibited lowest IC50 values in cardamonin, cardamonin-Cu(II) and cardamonin-Fe(II)-treated cells, we next explored the underlying mechanism of action in this cell. All three compounds were found to induce apoptotic cell death via the intrinsic mitochondrial pathway in Jurkat T cells as evidenced by the morphological changes, phosphatidylserine externalisation, caspase-3, -9 and PARP-1 cleavage, and collapse of mitochondrial membrane potential. Caspase-8, an initiator caspase of the extrinsic pathway was not activated. The presence of a caspase inhibitor, Z-VAD-FMK, was able to inhibit caspase processing and block cell death, further confirming the induced apoptotic cell death was caspase-dependent. As previous studies have reported the ability of metals to induce apoptosis through oxidative stress, the effects of these three compounds on reactive oxygen species (ROS) generation and intracellular glutathione (GSH) levels were explored. Results revealed cardamonin and cardamonin-Fe(II)-induced depletion of intracellular GSH and production of ROS; whereas cardamonin-Cu(II) did not significantly affect intracellular GSH levels but generated ROS. The presence of low molecular weight thiols, N-acetylcysteine (NAC), L-cysteine and GSH as well as Trolox, a ROS scavenger, blocked cardamonin and cardamonin-Fe(II)-induced Jurkat T cell death, while D-cysteine, which cannot be metabolised to GSH had no effect. However, these low molecular weight thiols had no effect on cardamonin-Cu(II)- treated cells with the exception of Trolox, confirming the role of ROS in cardamonin- Cu(II)-induced Jurkat T cell death. Furthermore, intracellular ROS and GSH levels were recovered close to levels of untreated Jurkat T cells in the presence of Z-VAD- FMK. In conclusion, this study demonstrates cardamonin, cardamonin-Cu(II) and cardamonin-Fe(II) induced apoptotic cell death in Jurkat T cells via an oxidative stress mediated intrinsic mitochondrial pathway that was caspase-dependent.

Dengue fever has emerged as one of the fastest growing health problems in recent years. Transmission of the mosquito-borne disease is widespread throughout the tropics, causing intriguing effects to the poorer populations with limited access to healthcare. At present, most vector control programmes have failed to contain the disease and no specific treatment is available yet. The existence of dengue virus as four distinct serotypes poses a significant threat as secondary infection is often manifested in a more severe form leading to hospitalisation and even death. Hence, this pushes the demand for a dengue vaccine as a long-term protective approach. Particularly for developing nations, cost is a major factor that needs to be meticulously addressed in order to provide a quick yet affordable medical relief. With that, this study aimed at producing a safe and cost-effective plant-based dengue subunit vaccine to protect against the febrile illness. A consensus sequence of the dengue envelope glycoprotein domain III (namely cEDIII) was selected as the antigenic determinant. cEDIII was expressed in two forms, i.e. as recombinant proteins with fusion to green fluorescent protein (sGFP) or cholera toxin B subunit (CTB), and as an epitope display on hepatitis B core antigen (HBcAg) virus-like particles (VLPs). All the constructs were cloned into pEAQ-HT vector and transient expression was achieved via agroinfiltration of Nicotiana benthamiana plants. Following the successful detection of heterologous proteins in N. benthamiana, purification procedures were carried out to harvest the recombinant proteins and chimeric HBcAg VLPs-displaying cEDIII, correspondingly. The recombinant fusion of cEDIII to CTB was shown to preserve the ability to fold into its active pentamer and bind with native gangliosides. Meanwhile, assembly of the chimeric HBcAg VLPs-displaying cEDIII was verified via transmission electron microscopy. These purified recombinant proteins and chimeric VLPs were then used for immunogenicity testing in BALB/c mice. Following vaccination with the recombinant protein, the results showed that successful production of anti-cEDIII specific response with neutralising potency against four dengue serotypes was obtained. T cell analyses suggested that the cEDIII induced a predominant T helper (Th) 1 response while the fusion with CTB could skew the response towards a mixed Th1/Th2. Immunisation with the chimeric VLPs-displaying cEDIII also achieved induction of cEDIII-specific responses, however, further evaluations are needed to warrant the successful use of these VLPs-based vaccines. Overall, the findings in this study have provided solid evidence that the development of a plant-based dengue vaccine is feasible. This is of crucial importance to battle against the upsurge of disease burden, and the production of a local vaccine could complement with Malaysian government’s efforts in combating dengue disease.