Tetrahydrobiopterin metabolism in dementia

The aim of this study was to establish levels of the enzymes involved in tetrahydrobiopterin (BH4) metabolism in human and rat brain preparations; to determine whether BH4 metabolism is altered in dementia, particularly in relation to senile dementia of the Alzheimer type (SDAT); and to examine the effect of aluminium on BH4 metabolism. Overall BH4 synthesis and dihydropteridine reductase (DHPR) activity were greater in the locus coeruleus than in the neocortex of elderly subjects. Sepiapterin reductase and DHPR activity showed a linear correlation with age in the temporal cortex. DHPR activity in the frontal cortex was relatively constant until the mid 60s and then fell with age. Overall BH4 synthesis showed a non-significant decline in temporal cortex and was significantly reduced in locus coeruleus preparations from SDAT subjects compared to control subjects. As DHPR, sepiapterin reductase and GTP cyclohydrolase activity were unaltered in SDAT we suggested that there is a lesion on the biosynthetic pathway between dihydroneopterin in triphosphate and BH4 in SDAT, possibly at the level of 6-pyruvoyl tetrahydropterin synthase. DHPR activity and BH4 synthesis capacity were unaltered in temporal cortex preparations from Huntingdon's disease subjects indicating that the defect in BH4 metabolism in SDAT is specific to the disease process and not a secondary consequence of dementia. The implications of altered BH4 metabolism in ageing and dementia are discussed. BH4 metabolism was examined in temporal and frontal cortex preparations from 4 subjects who had received peritoneal dialysis treatment. All patients had elevated serum aluminium levels. The data suggests that aluminium may inhibit DHPR activity in the frontal cortex resulting in diminished BH4 levels in the cells which leads to a compensatory increase in the activity of the biosynthetic pathway. Aluminium reversibly inhibited sepiapterin reductase activity in rat brain preparations but did not alter sepiapterin reductase activity in vivo. Overall BH4 synthesis and OTP cyclohydrolase activity were not affected by aluminium in vitro. The biosynthetic pathway was unaltered in rat brain preparations from animals receiving aluminium orally compared to control animals. DHPR activity was unaltered or increased in rat brain preparations from aluminium treated rats compared to the control group.

A dynamic model for the activated sludge treatment of coke oven effluents

This is an Inter-Disciplinary Higher Degree (IHD) thesis about Water Pollution Control in the Iron and Steel Industry. After examining the compositions, and various treatment methods, for the major effluent streams from a typical Integrated Iron and Steel works, it was decided to concentrate investigative work on the activated-sludge treatment of coke-oven effluents. A mathematical model of this process was developed in an attempt to provide a tool for plant management that would enable improved performance, and enhanced control of Works Units. The model differs from conventional models in that allowance is made for the presence of two genera of microorganisms, each of which utilises a particular type of substrate as its energy source. Allowance is also made for the inhibitive effect of phenol on thiocyanate biodegradation, and for the self-toxicity of the bacteria when present in a high substrate concentration environment. The enumeration of the kinetic characteristics of the two groups of micro-organisms was shown to be of major importance. Laboratory experiments were instigated in an attempt to determine accurate values of these coefficients. The use of the Suspended Solids concentration was found to be too insensitive a measure of viable active mass. Other measures were investigated, and Adenosine Triphosphate concentration was chosen as the most effective measure of bacterial populations. Using this measure, a model was developed for phenol biodegradation from experimental results which implicated the possibility of storage of substate prior to metabolism. A model for thiocyanate biodegradation was also developed, although the experimental results indicate that much work is still required in this area.

Vascular cell responsiveness to toll-like receptor ligands in carotid atheroma

Background Atherosclerosis is potentiated by stimulation of Toll-like receptors (TLRs), which serve to detect pathogen associated molecular patterns (PAMPs). However little is known of which PAMPs may be present in atheroma, or capable of stimulating inflammatory signalling in vascular cells. Materials and Methods DNA extracted from human carotid atheroma samples was amplified and sequenced using broad-range 16S gene specific primers to establish historical exposure to bacterial PAMPs. Responsiveness of primary human arterial and venous endothelial and smooth muscle cells to PAMPs specific for each of the TLRs was assessed by measurement of interleukin-8 secretion and E-selectin expression. Results Extracts of atheromatous tissue stimulated little or no signalling in TLR-transfected HEK-293 cells. However, sequencing of bacterial DNA amplified from carotid atheroma revealed the presence of DNA from 17 different bacterial genera, suggesting historical exposure to bacterial lipopeptide, lipopolysaccharide and flagellin. All cells examined were responsive to the ligands of TLR3 and TLR4, poly inosine:cytosine and lipopolysaccharide. Arterial cells were responsive to a wider range of PAMPs than venous cells, being additionally responsive to bacterial flagellin and unmethylated cytosine-phosphate-guanosine DNA motifs, the ligands of TLR5 and TLR9, respectively. Cells were generally unresponsive towards the ligands of human TLR7 and TLR8, loxoribine and single stranded RNA. Only coronary artery endothelial cells expressed TLR2 mRNA and responded to the TLR2 ligand Pam3CSK4. Conclusions Vascular cells are responsive to a relatively diverse range of TLR ligands and may be exposed, at least transiently, to ligands of TLR2, TLR4, TLR5 and TLR9 during the development of carotid atheroma.

Mechanistic differences between GSH transport by multidrug resistance protein 1 (MRP1/ABCC1) and GSH modulation of MRP1-mediated transport

Multidrug resistance protein 1 (MRP1/ABCC1) is an ATP-dependent polytopic membrane protein that transports many anticancer drugs and organic anions. Its transport mechanism is multifaceted, especially with respect to the participation of GSH. For example, vincristine is cotransported with GSH, estrone sulfate transport is stimulated by GSH, or MRP1 can transport GSH alone, and this can be stimulated by compounds such as verapamil or apigenin. Thus, the interactions between GSH and MRP1 are mechanistically complex. To examine the similarities and differences among the various GSH-associated mechanisms of MRP1 transport, we have measured first the effect of GSH and several GSH-associated substrates/modulators on the binding and hydrolysis of ATP by MRP1 using 8-azidoadenosine-5'-[(32)P]-triphosphate ([(32)P]azidoATP) analogs, and second the initial binding of GSH and GSH-associated substrates/modulators to MRP1. We observed that GSH or its nonreducing derivative S-methylGSH (S-mGSH), but none of the GSH-associated substrate/modulators, caused a significant increase in [gamma-(32)P]azidoATP labeling of MRP1. Moreover, GSH and S-mGSH decreased levels of orthovanadate-induced trapping of [alpha-(32)P]azidoADP. [alpha-(32)P]azidoADP.Vi trapping was also decreased by estone sulfate, whereas vincristine, verapamil, and apigenin had no apparent effects on nucleotide interactions with MRP1. Furthermore, estrone sulfate and S-mGSH enhanced the effect of each other 15- and 10-fold, respectively. Second, although GSH binding increased the apparent affinity of MRP1 for all GSH-associated substrates/modulators tested, only estrone sulfate had a reciprocal effect on the apparent affinity of MRP1 for GSH. Overall, these results indicate significant mechanistic differences between MRP1-mediated transport of GSH and the ability of GSH to modulate MRP1 transport.

Characterization of tissue transglutaminase 2 in macrophage function

Apoptosis is a highly regulated process that removes damaged or unwanted cells in vivo and defective clearance of apoptotic cells by macrophages has significant immunological implications. Tissue transglutaminase 2 (TG2) is a Ca2+-dependent protein cross linking enzyme known to play an important role in cell proliferation, differentiation, carcinogenesis, programmed death, and aging. TG2 as a guanosine triphosphate (GTP)-binding or GTP- hydrolyzing protein for mediating signal transduction and as a cell cycle regulator emphasized the importance of this enzyme in aging process. The ubiquitous presence of TG2 compared to the other organ-specific TGases has attracted special attention as a cellular aging device. TG2 activity and expression are known to increase in aging humans suggesting possible involvement in several age-related processes such as decrease in vascular compliance and increased stiffening of conduit arteries, cataract formation, Alzheimer's disease and senescent epidermal keratinocytes. Our work aims to characterize the role of TG2 and its partners (e.g. syndecan-4 and ß3 integrin) in macrophage function. THP-1 cell derived macrophage-like cells and primary human macrophages were analyzed for the expression and function of TG2. Macrophage-apoptotic cell interaction studies in the presence of TG2 inhibitors resulted in significant inhibition of interaction. Macrophage cell surface TG2 and, in particular, its cell surface cross linking activity was found to be crucial in apoptotic cell clearance. Syndecan-4 association with TG2 implies possible cooperation of these proteins and knockdown studies of syndecan-4 reveal its importance in apoptotic cell clearance. Our current findings suggest that TG2 has a crucial but yet to be fully defined role in apoptotic cell clearance.

Role of Ca2+ in proteolysis-inducing factor (PIF)-induced atrophy of skeletal muscle

Proteolysis-inducing factor (PIF) induces muscle loss in cancer cachexia through a high affinity membrane bound receptor. This study investigates the mechanism by which the PIF receptor communicates to intracellular signalling pathways. C2C12 murine myoblasts were used as a model using PIF purified from MAC16 tumours. Calcium imaging was determined using fura-4-acetoxymethyl ester (Fura-4-AM). PIF induced a rapid rise in Ca2 +i, which was completely attenuated by a anti-receptor antibody, or peptides representing 20 mers of the N-terminus of the PIF receptor. Other agents catabolic for skeletal muscle including angiotensin II (AngII) tumour necrosis factor-a (TNF-a) and lipopolysaccharide (LPS) also induced a rise in Ca2 +i, but this was not attenuated by anti-PIF-receptor antibody. The rise in Ca2 +i induced by PIF and AngII was completely attenuated by the Zn2 + chelator D-myo-inositol-1,2,6-triphosphate, and this was reversed by administration of exogenous Zn2 +. The Ca2 +i rise induced by PIF was independent of the presence of extracellular Ca2 +, and attenuated by the Ca2 + pump inhibitor thapsigargin, suggesting that the Ca2 +i rise was due to release from intracellular stores. This rise in Ca2 +i induced by PIF was attenuated by both the phospholipase C inhibitor U73122 and 2-APB, an inhibitor of the inositol 1,4,5-triphosphate receptor, suggesting the involvement of a G-protein. Binding of the PIF to its receptor in skeletal muscle triggers a rise in Ca2 +i, which initiates a signalling cascade leading to a depression in protein synthesis, and an increase in protein degradation.

Pharmacogenomic studies of the anticancer and immunosuppressive thiopurines mercaptopurine and azathioprine

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT • 6-Mercaptopurine (6-MP) and azathioprine (AZA) are both inactive prodrugs that require intracellular activation into the active 6-thioguanine nucleotides (6-TGNs). • This metabolic process undergoes three different competitive pathways that are catalysed by three different enzymes; xanthine oxidase (XO), thiopurine methyltransferase (TPMT) and inosine triphosphatase (ITPA), all of which exhibit genetic polymorphisms. • Although the impact of genetic variation in the TPMT gene on treatment outcome and toxicity has been demonstrated, the role of other polymorphisms remains less well known. WHAT THIS STUDY ADDS • New information on the allelic variation of these three enzymes (XO, TPMT and ITPA) and their influence on 6-MP/AZA metabolism and toxicity. • Confirmation of the association of TPMT polymorphism with haematological toxicity. • Identified potential genetic characteristics that may contribute to higher risk of adverse events (such as ITPA IVS2+21A→C mutation). AIMS - To examine the allelic variation of three enzymes involved in 6-mercaptopurine/azathioprine (6-MP/AZA) metabolism and evaluate the influence of these polymorphisms on toxicity, haematological parameters and metabolite levels in patients with acute lymphoblastic leukaemia (ALL) or inflammatory bowel disease (IBD). METHODS - Clinical data and blood samples were collected from 19 ALL paediatric patients and 35 IBD patients who were receiving 6-MP/AZA therapy. All patients were screened for seven genetic polymorphisms in three enzymes involved in mercaptopurine metabolism [xanthine oxidase, inosine triphosphatase (C94→A and IVS2+21A→C) and thiopurine methyltransferase]. Erythrocyte and plasma metabolite concentrations were also determined. The associations between the various genotypes and myelotoxicity, haematological parameters and metabolite concentrations were determined. RESULTS - Thiopurine methyltransferase variant alleles were associated with a preferential metabolism away from 6-methylmercaptopurine nucleotides (P = 0.008 in ALL patients, P = 0.038 in IBD patients) favouring 6-thioguanine nucleotides (6-TGNs) (P = 0.021 in ALL patients). Interestingly, carriers of inosine triphosphatase IVS2+21A→C variants among ALL and IBD patients had significantly higher concentrations of the active cytotoxic metabolites, 6-TGNs (P = 0.008 in ALL patients, P = 0.047 in IBD patients). The study confirmed the association of thiopurine methyltransferase heterozygosity with leucopenia and neutropenia in ALL patients and reported a significant association between inosine triphosphatase IVS2+21A→C variants with thrombocytopenia (P = 0.012). CONCLUSIONS - Pharmacogenetic polymorphisms in the 6-MP pathway may help identify patients at risk for associated toxicities and may serve as a guide for dose individualization.

Characterization of tissue transglutaminase in human osteoblast-like cells

Tissue transglutaminase (tTG) is a calcium-dependent and guanosine 5'-triphosphate (GTP) binding enzyme, which catalyzes the post-translational modification of proteins by forming intermolecular ε(ϒ-glutamyl)lysine cross-links. In this study, human osteoblasts (HOBs) isolated from femoral head trabecular bone and two osteosarcoma cell lines (HOS and MG-63) were studied for their expression and localization of tTG. Quantitative evaluation of transglutaminase (TG) activity determined using the [1,414C]-putrescine incorporation assay showed that the enzyme was active in all cell types. However, there was a significantly higher activity in the cell homogenates of MG-63 cells as compared with HOB and HOS cells (p <0.001). There was no significant difference between the activity of the enzyme in HOB and HOS cells. All three cell types also have a small amount of active TG on their surface as determined by the incorporation of biotinylated cadaverine into fibronectin. Cell surface-related tTG was further shown by preincubation of cells with tTG antibody, which led to inhibition of cell attachment. Western blot analysis clearly indicated that the active TG was tTG and immunocytochemistry showed it be situated in the cytosol of the cells. In situ extracellular enzyme activity also was shown by the cell-mediated incorporation of fluorescein cadaverine into extracellular matrix (ECM) proteins. These results clearly showed that MG-63 cells have high extracellular activity, which colocalized with the ECM protein fibronectin and could be inhibited by the competitive primary amine substrate putrescine. The contribution of tTG to cell surface/matrix interactions and to the stabilization of the ECM of osteoblast cells therefore could by an important factor in the cascade of events leading to bone differentiation and mineralization.

The topography of transmembrane segment six is altered during the catalytic cycle of P-glycoprotein

Structural evidence has demonstrated that P-glycoprotein (P-gp) undergoes considerable conformational changes during catalysis, and these alterations are important in drug interaction. Knowledge of which regions in P-gp undergo conformational alterations will provide vital information to elucidate the locations of drug binding sites and the mechanism of coupling. A number of investigations have implicated transmembrane segment six (TM6) in drug-P-gp interactions, and a cysteine-scanning mutagenesis approach was directed to this segment. Introduction of cysteine residues into TM6 did not disturb basal or drug-stimulated ATPase activity per se. Under basal conditions the hydrophobic probe coumarin maleimide readily labeled all introduced cysteine residues, whereas the hydrophilic fluorescein maleimide only labeled residue Cys-343. The amphiphilic BODIPY-maleimide displayed a more complex labeling profile. The extent of labeling with coumarin maleimide did not vary during the catalytic cycle, whereas fluorescein maleimide labeling of F343C was lost after nucleotide binding or hydrolysis. BODIPY-maleimide labeling was markedly altered during the catalytic cycle and indicated that the adenosine 5'-(beta,gamma-imino)triphosphate-bound and ADP/vanadate-trapped intermediates were conformationally distinct. Our data are reconciled with a recent atomic scale model of P-gp and are consistent with a tilting of TM6 in response to nucleotide binding and ATP hydrolysis.

The role of Ca2+ in the generation of spontaneous astrocytic Ca2+ oscillations

Astrocytes in the rat thalamus display spontaneous [Ca2+]i oscillations that are due to intracellular release, but are not dependent on neuronal activity. In this study we have investigated the mechanisms involved in these spontaneous [Ca2+]i oscillations using slices loaded with Fluo-4 AM (5 μM) and confocal microscopy. Bafilomycin A1 incubation had no effect on the number of spontaneous [Ca2+]i oscillations indicating that they were not dependent on vesicular neurotransmitter release. Oscillations were also unaffected by ryanodine. Phospholipase C (PLC) inhibition decreased the number of astrocytes responding to metabotropic glutamate receptor (mGluR) activation but did not reduce the number of spontaneously active astrocytes, indicating that [Ca2+]i increases are not due to membrane-coupled PLC activation. Spontaneous [Ca2+]i increases were abolished by an IP3 receptor antagonist, whilst the protein kinase C (PKC) inhibitor chelerythrine chloride prolonged their duration, indicating a role for PKC and inositol 1,4,5,-triphosphate receptor activation. BayK8644 increased the number of astrocytes exhibiting [Ca2+]i oscillations, and prolonged the responses to mGluR activation, indicating a possible effect on store-operated Ca2+ entry. Increasing [Ca2+]o increased the number of spontaneously active astrocytes and the number of transients exhibited by each astrocyte. Inhibition of the endoplasmic reticulum Ca2+ ATPase by cyclopiazonic acid also induced [Ca2+]i transients in astrocytes indicating a role for cytoplasmic Ca2+ in the induction of spontaneous oscillations. Incubation with 20 μM Fluo-4 reduced the number of astrocytes exhibiting spontaneous increases. This study indicates that Ca2+ has a role in triggering Ca2+ release from an inositol 1,4,5,-triphosphate sensitive store in astrocytes during the generation of spontaneous [Ca2+]i oscillations

Investigation of molecular mechanisms of LKB1 function in Dictyostelium development and stress responses and the function of superoxide dismutase C (SodC) in chemotaxis

The serine/threonine kinase LKB1 is a regulator of critical events including development and stress responses in metazoans. The current study was undertaken to determine the function of LKB1 in Dictyostelium . During multicellular development and in response to stress insult, an apparent increase in the DdLKB1 kinase activity was observed. Depletion of DdLKB1 with a knockdown construct led to aberrant development; a severe reduction in prespore cell differentiation and a precocious induction of prestalk cells, which were reminiscent of cells lacking GSK3, a well known cell-fate switch. Furthermore, DdLKB1 depleted cells displayed lower GSK3 activity than wild type cells in response to cAMP stimulation during development and failed to activate AMPK, a well known LKB1 target in mammals, in response to cAMP and stress insults. These results suggest that DdLKB1 positively regulates both GSK3 and AMPK during Dictyostelium development, and DdLKB1 is necessary for AMPK activation during stress response regulation. No apparent GSK3 activation was observed in response to stress insults. Spatial and temporal regulation of phosphatidylinositol-(3,4,5)-triphosphate (PIP3) along the membrane of polarized cells is important for efficient chemotaxis. A REMI screen for PIP3 suppressors in the absence of stimulation led to the identification of SodC as PIP3 regulator. Consistent with their higher PIP3 levels, sodC− cells showed defects in chemotaxis and exhibited higher intra-cellular superoxide levels. Protein localization studies along with observations from GPI specific PI-PLC treatment of wild-type cells suggested that SodC is a GPI anchored outer-membrane protein. SodC showed superoxide dismutase activity in vitro, and motility defects of sodC− cells can be rescued by expressing the intact SodC but not by the mutant SodC, which has point mutations that affect its dismutase function. Treatment of sodC− cells with LY294002, a pharmacological inhibitor of PI3K, partially rescued the polarization and chemoattractant sensing defects but not motility defects. Consistent with increased intracellular superoxide levels, sodC − cells also exhibited higher basal Ras activity, an upstream regulator of PI3K, which can be suppressed by a cell permeable superoxide scavenger, XTT, indicating that SodC is important in regulation of intracellular superoxide levels thereby regulating the Ras activity and PIP3 levels at the membrane.

Glycogen Synthase Kinase 3 influences cell motility and chemotaxis by regulating phosphatidylinositol 3 kinase localization in Dictyostelium discoideum

Glycogen Synthase Kinase 3 (GSK3), a serine/threonine kinase initially characterized in the context of glycogen metabolism, has been repeatedly realized as a multitasking protein that can regulate numerous cellular events in both metazoa and protozoa. I recently found GSK3 plays a role in regulating chemotaxis, a guided cell movement in response to an external chemical gradient, in one of the best studied model systems for chemotaxis - Dictyostelium discoideum. ^ It was initially found that comparing to wild type cells, gsk3 - cells showed aberrant chemotaxis with a significant decrease in both speed and chemotactic indices. In Dictyostelium, phosphatidylinositol 3,4,5-triphosphate (PIP3) signaling is one of the best characterized pathways that regulate chemotaxis. Molecular analysis uncovered that gsk3- cells suffer from high basal level of PIP3, the product of PI3K. Upon chemoattractant cAMP stimulation, wild type cells displayed a transient increase in the level of PIP3. In contrast, gsk3- cells exhibited neither significant increase nor adaptation. On the other hand, no aberrant dynamic of phosphatase and tensin homolog (PTEN), which antagonizes PI3K function, was observed. Upon membrane localization of PI3K, PI3K become activated by Ras, which will in turn further facilitate membrane localization of PI3K in an F-Actin dependent manner. The gsk3- cells treated with F-Actin inhibitor Latrunculin-A showed no significant difference in the PIP3 level. ^ I also showed GSK3 affected the phosphorylation level of the localization domain of PI3K1 (PI3K1-LD). PI3K1-LD proteins from gsk3- cells displayed less phosphorylation on serine residues compared to that from wild type cells. When the potential GSK3 phosphorylation sites of PI3K1-LD were substituted with aspartic acids (Phosphomimetic substitution), its membrane localization was suppressed in gsk3- cells. When these serine residues of PI3K1-LD were substituted with alanine, aberrantly high level of membrane localization of the PI3K1-LD was monitored in wild type cells. Wild type, phosphomimetic, and alanine substitution of PI3K1-LD fused with GFP proteins also displayed identical localization behavior as suggested by the cell fraction studies. Lastly, I identified that all three potential GSK3 phosphorylation sites on PI3K1-LD could be phosphorylated in vitro by GSK3.^

Theoretical Investigation of Intra- and Inter-cellular Spatiotemporal Calcium Patterns in Microcirculation

Microcirculatory vessels are lined by endothelial cells (ECs) which are surrounded by a single or multiple layer of smooth muscle cells (SMCs). Spontaneous and agonist induced spatiotemporal calcium (Ca2+) events are generated in ECs and SMCs, and regulated by complex bi-directional signaling between the two layers which ultimately determines the vessel tone. The contractile state of microcirculatory vessels is an important factor in the determination of vascular resistance, blood flow and blood pressure. This dissertation presents theoretical insights into some of the important and currently unresolved phenomena in microvascular tone regulation. Compartmental and continuum models of isolated EC and SMC, coupled EC-SMC and a multi-cellular vessel segment with deterministic and stochastic descriptions of the cellular components were developed, and the intra- and inter-cellular spatiotemporal Ca2+ mobilization was examined. Coupled EC-SMC model simulations captured the experimentally observed localized subcellular EC Ca2+ events arising from the opening of EC transient receptor vanilloid 4 (TRPV4) channels and inositol triphosphate receptors (IP3Rs). These localized EC Ca2+ events result in endothelium-derived hyperpolarization (EDH) and Nitric Oxide (NO) production which transmit to the adjacent SMCs to ultimately result in vasodilation. The model examined the effect of heterogeneous distribution of cellular components and channel gating kinetics in determination of the amplitude and spread of the Ca2+ events. The simulations suggested the necessity of co-localization of certain cellular components for modulation of EDH and NO responses. Isolated EC and SMC models captured intracellular Ca2+ wave like activity and predicted the necessity of non-uniform distribution of cellular components for the generation of Ca2+ waves. The simulations also suggested the role of membrane potential dynamics in regulating Ca2+ wave velocity. The multi-cellular vessel segment model examined the underlying mechanisms for the intercellular synchronization of spontaneous oscillatory Ca2+ waves in individual SMC. From local subcellular events to integrated macro-scale behavior at the vessel level, the developed multi-scale models captured basic features of vascular Ca2+ signaling and provide insights for their physiological relevance. The models provide a theoretical framework for assisting investigations on the regulation of vascular tone in health and disease.

Investigation of molecular mechanisms of lkb1 function in dictyostelium development and stress responses and the function of superoxide dismutase c (sodc) in chemotaxis

The serine/threonine kinase LKB1 is a regulator of critical events including development and stress responses in metazoans. The current study was undertaken to determine the function of LKB1 in Dictyostelium. During multicellular development and in response to stress insult, an apparent increase in the DdLKB1 kinase activity was observed. Depletion of DdLKB1 with a knockdown construct led to aberrant development; a severe reduction in prespore cell differentiation and a precocious induction of prestalk cells, which were reminiscent of cells lacking GSK3, a well known cell-fate switch. Furthermore, DdLKB1 depleted cells displayed lower GSK3 activity than wild type cells in response to cAMP stimulation during development and failed to activate AMPK, a well known LKB1 target in mammals, in response to cAMP and stress insults. These results suggest that DdLKB1 positively regulates both GSK3 and AMPK during Dictyostelium development, and DdLKB1 is necessary for AMPK activation during stress response regulation. No apparent GSK3 activation was observed in response to stress insults. Spatial and temporal regulation of phosphatidylinositol-(3,4,5)-triphosphate (PIP3) along the membrane of polarized cells is important for efficient chemotaxis. A REMI screen for PIP3 suppressors in the absence of stimulation led to the identification of SodC as PIP3 regulator. Consistent with their higher PIP3 levels, sodC- cells showed defects in chemotaxis and exhibited higher intra-cellular superoxide levels. Protein localization studies along with observations from GPI specific PI-PLC treatment of wild-type cells suggested that SodC is a GPI anchored outer-membrane protein. SodC showed superoxide dismutase activity in vitro, and motility defects of sodC- cells can be rescued by expressing the intact SodC but not by the mutant SodC, which has point mutations that affect its dismutase function. Treatment of sodC- cells with LY294002, a pharmacological inhibitor of PI3K, partially rescued the polarization and chemoattractant sensing defects but not motility defects. Consistent with increased intracellular superoxide levels, sodC- cells also exhibited higher basal Ras activity, an upstream regulator of PI3K, which can be suppressed by a cell permeable superoxide scavenger, XTT, indicating that SodC is important in regulation of intracellular superoxide levels thereby regulating the Ras activity and PIP3 levels at the membrane.

Bulk sediment characteristics and geomicrobiology of sediment core 5022-1J from Laguna Potrok Aike, southern Patagonia

Living microorganisms inhabit every environment of the biosphere but only in the last decades their importance governing biochemical cycles in deep sediments has been widely recognized. Most investigations have been accomplished in the marine realm whereas there is a clear paucity of comparable studies in lacustrine sediments. One of the main challenges is to define geomicrobiological proxies that can be used to identify different microbial signals in the sediments. Laguna Potrok Aike, a maar lake located in Southeastern Patagonia, has an annually not stratifying cold water column with temperatures ranging between 4 and 10 °C, and most probably an anoxic water/sediment interface. These unusual features make it a peculiar and interesting site for geomicrobiological studies. Living microbial activity within the sediments was inspected by the first time in a sedimentary core retrieved during an ICDP-sponsored drilling operation. The main goals to study this cold subsaline environment were to characterize the living microbial consortium; to detect early diagenetic signals triggered by active microbes; and to investigate plausible links between climate and microbial populations. Results from a meter long gravity core suggest that microbial activity in lacustrine sediments can be sustained deeper than previously thought due to their adaptation to both changing temperature and oxygen availability. A multi-proxy study of the same core allowed defining past water column conditions and further microbial reworking of the organic fraction within the sediments. Methane content shows a gradual increase with depth as a result of the fermentation of methylated substrates, first methanogenic pathway to take place in the shallow subsurface of freshwater and subsaline environments. Statistical analyses of DGGE microbial diversity profiles indicate four clusters for Bacteria reflecting layered communities linked to the oxidant type whereas three clusters characterize Archaea communities that can be linked to both denitrifiers and methanogens. Independent sedimentary and biological proxies suggest that organic matter production and/or preservation have been lower during the Medieval Climate Anomaly (MCA) coinciding with a low microbial colonization of the sediments. Conversely, a reversed trend with higher organic matter content and substantial microbial activity characterizes the sediments deposited during the Little Ice Age (LIA). Thus, the initial sediments deposited during distinctive time intervals under contrasting environmental conditions have to be taken into account to understand their impact on the development of microbial communities throughout the sediments and their further imprint on early diagenetic signals.