|
| ||
|
|
||
Biochemical Pathways (biochemical + pathway)
Selected AbstractsMetabolic engineering of Saccharomyces cerevisiae for the synthesis of the wine-related antioxidant resveratrolFEMS YEAST RESEARCH, Issue 1 2003John V.W. Becker Abstract The stilbene resveratrol is a stress metabolite produced by Vitis vinifera grapevines during fungal infection, wounding or UV radiation. Resveratrol is synthesised particularly in the skins of grape berries and only trace amounts are present in the fruit flesh. Red wine contains a much higher resveratrol concentration than white wine, due to skin contact during fermentation. Apart from its antifungal characteristics, resveratrol has also been shown to have cancer chemopreventive activity and to reduce the risk of coronary heart disease. It acts as an antioxidant and anti-mutagen and has the ability to induce specific enzymes that metabolise carcinogenic substances. The objective of this pilot study was to investigate the feasibility of developing wine yeasts with the ability to produce resveratrol during fermentation in both red and white wines, thereby increasing the wholesomeness of the product. To achieve this goal, the phenylpropanoid pathway in Saccharomyces cerevisiae would have to be introduced to produce p -coumaroyl-CoA, one of the substrates required for resveratrol synthesis. The other substrate for resveratrol synthase, malonyl-CoA, is already found in yeast and is involved in de novo fatty-acid biosynthesis. We hypothesised that production of p -coumaroyl-CoA and resveratrol can be achieved by co-expressing the coenzyme-A ligase-encoding gene (4CL216) from a hybrid poplar and the grapevine resveratrol synthase gene (vst1) in laboratory strains of S. cerevisiae. This yeast has the ability to metabolise p -coumaric acid, a substance already present in grape must. This compound was therefore added to the synthetic media used for the growth of laboratory cultures. Transformants expressing both the 4CL216 and vst1 genes were obtained and tested for production of resveratrol. Following ,-glucosidase treatment of organic extracts for removal of glucose moieties that are typically bound to resveratrol, the results showed that the yeast transformants had produced the resveratrol ,-glucoside, piceid. This is the first report of the reconstruction of a biochemical pathway in a heterologous host to produce resveratrol. [source] Genome-wide analysis of gene expression in adult Anopheles gambiaeINSECT MOLECULAR BIOLOGY, Issue 1 2006O. Marinotti Abstract With their genome sequenced, Anopheles gambiae mosquitoes now serve as a powerful tool for basic research in comparative, evolutionary and developmental biology. The knowledge generated by these studies is expected to reveal molecular targets for novel vector control and pathogen transmission blocking strategies. Comparisons of gene-expression profiles between adult male and nonblood-fed female Anopheles gambiae mosquitoes revealed that roughly 22% of the genes showed sex-dependent regulation. Blood-fed females switch the majority of their metabolism to blood digestion and egg formation within 3 h after the meal is ingested, in detriment to other activities such as flight and response to environment stimuli. Changes in gene expression are most evident during the first, second and third days after a blood meal, when as many as 50% of all genes showed significant variation in transcript accumulation. After laying the first cluster of eggs (between 72 and 96 h after the blood meal), mosquitoes return to a nongonotrophic stage, similar but not identical to that of 3-day-old nonblood-fed females. Ageing and/or the nutritional state of mosquitoes at 15 days after a blood meal is reflected by the down-regulation of ,5% of all genes. A full description of the large number of genes regulated at each analysed time point and each biochemical pathway or biological processes in which they are involved is not possible within the scope of this contribution. Therefore, we present descriptions of groups of genes displaying major differences in transcript accumulation during the adult mosquito life. However, a publicly available searchable database (http://www.angagepuci.bio.uci.edu/) has been made available so that detailed analyses of specific groups of genes based on their descriptions, functions or levels of gene expression variation can be performed by interested investigators according to their needs. [source] Effect of prophenoloxidase expression knockout on the melanization of microfilariae in the mosquito Armigeres subalbatusINSECT MOLECULAR BIOLOGY, Issue 4 2001S. H. Shiao Abstract Melanization is an effective defence reaction used by mosquito hosts to kill malarial and filarial worm parasites. Although phenoloxidase (PO) has long been considered to be the key enzyme in the biosynthesis of melanotic material in insects, there is no direct evidence verifying its role in parasite melanization. To elucidate the role of PO in the melanization of microfilariae (mf) by mosquitoes, a double subgenomic Sindbis (dsSIN) recombinant virus was used to transduce Armigeres subalbatus mosquitoes with a 600 base antisense RNA targeted to the highly conserved copper-binding region of an Ar. subalbatus PO gene. Compared with controls, haemolymph PO activity in mosquitoes transduced with antisense RNA was significantly reduced. When these mosquitoes were challenged with Dirofilaria immitis mf, the melanization of mf was almost completely inhibited. These data verify that PO is an essential component of the biochemical pathway required for the melanization of parasites, and that the dsSIN expression system represents a useful tool in the functional analysis of endogenous gene expression in mosquitoes. [source] Correlations between the Sonic Hedgehog Pathway and basal cell carcinomaINTERNATIONAL JOURNAL OF DERMATOLOGY, Issue 11 2007Omar Lupi MD The Hedgehog (HH) family of intercellular signaling proteins has some essential functions in patterning both invertebrate and vertebrate embryos. Identified as an important regulator of segment polarity and tissue organization in flies, the HH pathway can also play a significant role in human development and in cutaneous carcinogenesis. The family received their name because when the D. melanogaster HH protein malfunctions the mutant fly ends up looking like a small prickly ball, similar to a curled up hedgehog. The Sonic hedgehog (SHH) pathway is implicated in the etiology of the most common human cancer, the basal cell carcinoma (BCC). Mutations in the receptor of SHH, the patched gene (PTCH), have been characterized in sporadic BCCs as well as those from patients with the rare genetic syndrome nevoid BCC. Human PTCH is mutated in sporadic as well as hereditary BCCs, and inactivation of this gene is probably a necessary if not sufficient step for tumorigenesis. Delineation of the biochemical pathway in which PTCH functions may lead to rational medical therapy for skin cancer and possibly other tumors. [source] Melanin biosynthesis by Frankia strain CeI5PHYSIOLOGIA PLANTARUM, Issue 2 2007Wenlin Yuan Many Frankia strains are pigmented and presumed to produce melanin. However, melanin biosynthesis has yet to be rigorously characterized in Frankia. This study was initiated to determine whether or not Frankia strain CeI5 produced melanin and to identify the biochemical pathway of pigment production. Frankia strain CeI5 first produced a dark pigment in mycelial and other tissue and then in the liquid culture medium when grown in a defined medium containing l -tyrosine. The pigment resisted solvents, lightened when subjected to the action of oxidants, as well as reductants, and produced a flocculent brown precipitate with FeCl3. Spectroscopic characteristics of the extracted pigment were those of melanin. When subjected to gradual dilution, the absorbance decreased unevenly, occurring in the near red range first, then in the visible range, and lastly in the UV range. This observation might resolve the question of why quite different descriptions of melanin UV,visible light absorption spectra exist in the literature. The tyrosinase cofactor copper greatly enhanced melanin biosynthesis at 5.3 × 10,6 M, while 1 × 10,8 M 3,4-dihydroxy- l -phenylalanine hastened pigmentation. The copper-chelating agent KCN and the tyrosinase inhibitor tropolone decreased melanin production at the same concentration of 1 × 10,5 M. This evidence suggests that Frankia strain CeI5 produces melanin via the Raper and Mason pathway. [source] Orange, yellow and white-cream: inheritance of carotenoid-based colour in sunflower pollenPLANT BIOLOGY, Issue 1 2010M. Fambrini Abstract Inheritance of pollen colour was studied in sunflower (Helianthus annuus L.) using three distinct pollen colour morphs: orange, yellow and white-cream. Orange is the most common colour of sunflower pollen, while the yellow morph is less frequent. These two types were observed in the inbred lines F11 and EF2L, respectively. White-cream pollen is a rare phenotype in nature, and was identified in a mutant, named white-cream pollen, recovered in the R2 generation of an in vitro regenerated plant. The F11 inbred line was used as starting material for in vitro regeneration. The carotenoid content of these three pollen morphs differed, and was extremely reduced in white-cream pollen. The phenotype of F1 populations obtained by reciprocal crosses revealed that the orange trait was dominant over both white-cream and yellow. Segregation of F2 populations of both crosses, orange × yellow and orange × white-cream, approached a 3:1 ratio, indicating the possibility of simple genetic control. By contrast, a complementation cross between the two lines with white-cream and yellow pollen produced F1 plants with orange pollen. The F2 populations of this cross-segregated as nine orange: four white-cream: four yellow. A model conforming to the involvement of two unlinked genes, here designated Y and O, can explain these results. Accessions with yellow pollen would have the genotype YYoo, the white-cream pollen mutant would have yyOO and the accession with orange pollen would have YYOO. Within F2 populations of the cross white-cream × yellow a new genotype, yyoo, with white-cream pollen was scored. The results of the cross yyoo × YYoo produced only F1 plants with yellow pollen, supporting a recessive epistatic model of inheritance between two loci. In this model, yy is epistatic on O and o. In F2 populations, the distributions of phenotypic classes suggested that the genetic control of carotenoid content is governed by major genes, with large effects segregating in a background of polygenic variation. These three pollen morphs can provide insight into the sequence in which genes act, as well into the biochemical pathway controlling carotenoid biosynthesis in anthers and the transfer of these different pigments into pollenkitt. [source] SPLASH: Systematic proteomics laboratory analysis and storage hubPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 6 2006Siaw Ling Lo Abstract In the field of proteomics, the increasing difficulty to unify the data format, due to the different platforms/instrumentation and laboratory documentation systems, greatly hinders experimental data verification, exchange, and comparison. Therefore, it is essential to establish standard formats for every necessary aspect of proteomics data. One of the recently published data models is the proteomics experiment data repository [Taylor, C. F., Paton, N. W., Garwood, K. L., Kirby, P. D. et,al., Nat. Biotechnol. 2003, 21, 247,254]. Compliant with this format, we developed the systematic proteomics laboratory analysis and storage hub (SPLASH) database system as an informatics infrastructure to support proteomics studies. It consists of three modules and provides proteomics researchers a common platform to store, manage, search, analyze, and exchange their data. (i),Data maintenance includes experimental data entry and update, uploading of experimental results in batch mode, and data exchange in the original PEDRo format. (ii),The data search module provides several means to search the database, to view either the protein information or the differential expression display by clicking on a gel image. (iii),The data mining module contains tools that perform biochemical pathway, statistics-associated gene ontology, and other comparative analyses for all the sample sets to interpret its biological meaning. These features make SPLASH a practical and powerful tool for the proteomics community. [source] Structure of CDP- d -glucose 4,6-dehydratase from Salmonella typhi complexed with CDP- d -xyloseACTA CRYSTALLOGRAPHICA SECTION D, Issue 4 2005Nicole M. Koropatkin Tyvelose is a unique 3,6-dideoxyhexose found in the O antigens of some pathogenic species of Yersinia and Salmonella. It is produced via a complex biochemical pathway that employs CDP- d -glucose as the starting ligand. CDP- d -glucose 4,6-dehydratase catalyzes the first irreversible step in the synthesis of this 3,6-dideoxysugar by converting CDP- d -glucose to CDP-4-keto-6-deoxyglucose via an NAD+ -dependent intramolecular oxidation,reduction reaction. Here, the cloning, protein purification and X-ray crystallographic analysis of CDP- d -glucose 4,6-dehydratase from Salmonella typhi complexed with the substrate analog CDP- d -xylose are described. Each subunit of the tetrameric enzyme folds into two domains. The N-terminal region contains a Rossmann fold and provides the platform for NAD(H) binding. The C-terminal motif is primarily composed of ,-helices and houses the binding pocket for the CDP portion of the CDP- d -xylose ligand. The xylose moiety extends into the active-site cleft that is located between the two domains. Key residues involved in anchoring the sugar group to the protein include Ser134, Tyr159, Asn197 and Arg208. Strikingly, Ser134,O, and Tyr159,O, sit within 2.9,Ĺ of the 4,-hydroxyl group of xylose. Additionally, the side chains of Asp135 and Lys136 are located at 3.5 and 3.2,Ĺ, respectively, from C-5 of xylose. In the structurally related dTDP- d -glucose 4,6-dehydratase, the Asp/Lys pair is replaced with an Asp/Glu couple. On the basis of this investigation, it can be speculated that Tyr159 serves as the catalytic base to abstract the 4,-hydroxyl proton from the sugar and that Asp135 and Lys136 play critical roles in the subsequent dehydration step that leads to the final product. [source] Depression gets old fast: do stress and depression accelerate cell aging?,DEPRESSION AND ANXIETY, Issue 4 2010Owen M. Wolkowitz M.D. Abstract Depression has been likened to a state of "accelerated aging," and depressed individuals have a higher incidence of various diseases of aging, such as cardiovascular and cerebrovascular diseases, metabolic syndrome, and dementia. Chronic exposure to certain interlinked biochemical pathways that mediate stress-related depression may contribute to "accelerated aging," cell damage, and certain comorbid medical illnesses. Biochemical mediators explored in this theoretical review include the hypothalamic,pituitary,adrenal axis (e.g., hyper- or hypoactivation of glucocorticoid receptors), neurosteroids, such as dehydroepiandrosterone and allopregnanolone, brain-derived neurotrophic factor, excitotoxicity, oxidative and inflammatory stress, and disturbances of the telomere/telomerase maintenance system. A better appreciation of the role of these mediators in depressive illness could lead to refined models of depression, to a re-conceptualization of depression as a whole body disease rather than just a "mental illness," and to the rational development of new classes of medications to treat depression and its related medical comorbidities. Depression and Anxiety, 2010. © 2010 Wiley-Liss, Inc. [source] Monoclonal antibodies: a morphing landscape for therapeuticsDRUG DEVELOPMENT RESEARCH, Issue 10 2006Nicholas C. Nicolaides Abstract The concept of using antibodies as therapeutics to cure human diseases was postulated nearly 100 years ago by Paul Ehrlich and subsequently enabled by the discovery of hybridoma technology by Kohler and Milstein in 1975. While the use of monoclonal antibodies (mAbs) as drugs that can specifically target a disease-associated antigen is compelling, it has taken a quarter century for these molecules to be adopted as bona fide therapeutic agents. Despite their slow pursuit in drug development during the pioneering years, it is now estimated that there are nearly 500 mAb-based therapies in development. Major factors that have influenced the acceptance of monoclonal antibodies as therapeutics include their drug safety profiles, technological advancements for facilitating mAb discovery and development, and market success. Early on, it was demonstrated that antibodies could elicit clinical benefit by antagonizing a specific antigen without the common side effects that are prevalent with small chemical entities due to their nonspecific effects on homeostatic biochemical pathways. In addition, the significant technological advances that the biotechnology industry has established for developing and producing monoclonal antibodies at commercial scale in a more efficient and cost-effective manner has broadly enabled their use as therapeutics. However, despite the beneficial pharmacologic advantages and technological advances, it has been the sheer market success that monoclonal antibody products have achieved over the past few years that has propelled their vast pursuit by the biopharmaceutical industry in light of their value-creating potential. Here we provide an overview of the monoclonal antibody industry and discuss evolving technologies and strategies that are being pursued to overcome challenges in the changing marketplace. Drug Dev. Res. 67:781,789, 2006. © 2007 Wiley-Liss, Inc. [source] The autocorrelation matrix probing biochemical relationships after metabolic fingerprinting with CEELECTROPHORESIS, Issue 7 2009Santiago Angulo Abstract Fingerprinting together with statistical analysis is often employed to compare samples in metabonomic studies of a disease. Correlation algorithms can aid by extracting information based on the variation patterns of key metabolites. This information can be linked to metabolite identification or to specific up/down-regulated biochemical pathways. Matlab-based software employing the Pearson's correlation algorithm was applied to urine electropherograms from 20 mice infected with the schistosoma parasite. The fingerprints were the sum of electropherograms analysed with normal and reverse polarity, in two different modes MEKC and CZE and with two different capillaries (uncoated and polyacrylamide coated) to provide a broad picture of the samples. Hippurate, a metabolite that was depleted in the infected group and is present in both polarities, was chosen as a test variable; it correlated with itself to a p value of <0.000. Phenylacetylglycine, a metabolite shown as over expressed in the disease, was positively correlated to three metabolites in its same pathway with a correlation coefficient of 0.7 and p<0.000 to phenylalanine, 0.7 and p<0.000 to 2-hydroxyphenylacetic and 0.55 and p<0.003 to phenylacetate. The study shows that the autocorrelation matrix is able to provide extra information from data files acquired by CE analyses. It underlined an up-regulated metabolic path by association in the schistosoma infection model. [source] Proteomic mapping of the hyperthermophilic and acidophilic archaeon Sulfolobus solfataricus P2ELECTROPHORESIS, Issue 14 2006Richard C. Barry Abstract A proteomic map of Sulfolobus solfataricus,P2, an archaeon that grows optimally at 80°C and pH,3.2, was developed using high-resolution 2-DE and peptide mass fingerprinting. A total of 867,protein spots (659,aqueous Tris-soluble spots and 208,aqueous Tris-insoluble) were mapped over IPG,3,10, 4,7, and 6,11, with second-dimensional gels made of 8,18%,polyacrylamide. Three hundred and twenty-four different gene products were represented by the 867,spots, with 274,gene products being identified in the Tris-soluble fractions and 100,gene products in the Tris-insoluble portion. Fifty gene products were found on gels from both fractions. Additionally, an average of 1.50 ± 0.12 isoforms/protein was identified. This mapping study confirmed the expression of proteins involved in numerous metabolic, transport, energy production, nucleic acid replication, translation, and transcription pathways. Of particular interest, phosphoenolpyruvate carboxykinase,(SSO2537) was detected even though the pathway for gluconeogenesis is unknown for this archaeon. Tris-soluble fractions contained many cytosolic proteins while Tris-insoluble fractions contained many membrane-associated proteins, including ABC transporters and an ATP synthase. This study provides an optimized 2-DE approach for investigating the biochemical pathways and post-translational modifications employed by Sulfolobus to survive in its extreme environment. [source] Complexes of glutathione with heavy metal ions as a new biochemical marker of aquatic environment pollution,ENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 3 2010Jiri Baloun Abstract Reduced glutathione (GSH) plays a number of key roles in many biochemical pathways. This peptide is highly reactive and forms conjugates with other molecules via its sulfhydryl moiety. The interactions of the common heavy metal pollutant Cd(II) with GSH were determined by using the Brdicka reaction to evaluate whether this technique would be suitable as a biomarker. After GSH interaction with Cd(II) ions, two characteristic changes in the measured voltammogram were observed: Cat2 signal height decreased, and a new signal called P1 was found. The observed signal probably relates to the formation of a GSH,heavy metal ion complex adsorbed on the surface of the working electrode. When the interaction of GSH with cisplatin was studied, the same characteristic changes in the voltammogram were observed, which confirmed our hypothesis. Moreover, changes in the height of P1 and Cat2 signals with increasing time of GSH interaction with Cd(II) ions and/or cisplatin were also investigated. Cat2 peak height decreased proportionally with increasing time of interaction. This decrease can be explained by shielding of free sulfhydryl moiety by heavy metal ions, so it cannot catalyze the evolution of hydrogen from the supporting electrolyte. In addition, we found that, with increasing time of the interaction, the P1 signal was enhanced and shifted to more positive potentials for both Cd(II) ions and cisplatin. Environ. Toxicol. Chem. 2010;29:497,500. © 2009 SETAC [source] Dose-dependent stimulation of hepatic retinoic acid hydroxylation/oxidation and glucuronidation in brook trout, Salvelinus fontinalis, after exposure to 3,3,,4,4,-tetrachlorobiphenylENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 3 2000Patrick M. Boyer Abstract Extremely low stores of vitamin A have been reported in fish and birds inhabiting regions contaminated by coplanar polychlorinated biphenyls (PCBs) and other organochlorines, suggesting many possible effects on retinoid biochemical pathways. Metabolic imbalances associated with biologically active retinoids (e.g., retinoic acid) could be associated with teratogenesis, edema, growth inhibition, reproductive impairment, immunosuppression, and susceptibility to cancer. Sexually mature brook trout were injected intraperitoneally with the coplanar PCB 3,3,,4,4,-tetrachlorobiphenyl (TCBP) and again 4 weeks later. At 8 weeks, retinoic acid metabolism was measured in liver microsomes. To our knowledge, retinoic acid conjugation by UDP-glucuronyltransferase is described here for the first time in fish. A substantial rate of glucuronidation was detected in the microsomes from control brook trout, which tended to increase over the dose range of TCBP. Glucuronidation was significantly greater in fish receiving the 10 ,g/g body weight dose level. Metabolism through the cytochrome P450 system was also dose-dependent, resulting in significantly greater production of 4-hydroxyretinoic acid at the 10 ,g/g dose level. In contrast, subsequent oxidation to 4- oxo -retinoic acid was greatest at the 1 ,g/g dose level and did not increase further at higher doses. Liver stores of dehydroretinyl palmitate/oleate were significantly decreased at the 5 and 10 ,g/g dose levels. [source] Nitrifier denitrification can be a source of N2O from soil: a revised approach to the dual-isotope labelling methodEUROPEAN JOURNAL OF SOIL SCIENCE, Issue 5 2010D. M. Kool Nitrifier denitrification (i.e. nitrite reduction by ammonia oxidizers) is one of the biochemical pathways of nitrous oxide (N2O) production. It is increasingly suggested that this pathway may contribute substantially to N2O production in soil, the major source of this greenhouse gas. However, although monoculture studies recognize its potential, methodological drawbacks prohibit conclusive proof that nitrifier denitrification occurs in actual soils. Here we suggest and apply a new isotopic approach to identify its presence in soil. In incubation experiments with 12 soils, N2O production was studied using oxygen (O) and nitrogen (N) isotope tracing, accounting for O exchange. Microbial biomass C and N and phospholipid fatty acid (PLFA) patterns were analysed to explain potential differences in N2O production pathways. We found that in at least five of the soils nitrifier denitrification must have contributed to N2O production. Moreover, it may even have been responsible for all NH4+ -derived N2O in most soils. In contrast, N2O as a by-product of ammonia oxidation contributed very little to total production. Microbial biomass C and N and PLFA-distinguished microbial community composition were not indicative of differences in N2O production pathways. Overall, we show that combined O and N isotope tracing may still provide a powerful tool to understand N2O production pathways, provided that O exchange is accounted for. We conclude that nitrifier denitrification can indeed occur in soils, and may in fact be responsible for the greater proportion of total nitrifier-induced N2O production. [source] Hypotheses for the origin and early evolution of triterpenoid cyclasesGEOBIOLOGY, Issue 1 2007W. W. FISCHER ABSTRACT Hopanes and steranes are found almost universally in the sedimentary rock record where they often are used as proxies for aerobic organisms, metabolisms, and environments. In order to interpret ancient lipid signatures confidently we require a complementary understanding of how these modern biochemical pathways evolved since their conception. For example, generally it has been assumed that hopanoid biosynthesis was an evolutionary predecessor to steroid biosynthesis. Here we re-evaluate this assumption. Using a combined phylogenetic and biochemical perspective, we address the evolution of polycyclic triterpenoid biosynthesis and suggest several constraints on using these molecules as aerobic biomarkers. Amino acid sequence data show that the enzymes responsible for polycyclic triterpenoid biosynthesis (i.e. squalene and 2,3-oxidosqualene cyclases) are homologous. Numerous conserved domains correspond to active sites in the enzymes that are required to complete the complex cyclization reaction. From these sites we develop an evolutionary analysis of three independent characters to explain the evolution of the major classes of polycyclic triterpenoids. These characters are: (i) the number of unfavourable anti-Markovnikov ring closures, (ii) all-chair (CCC) or chair-boat-chair (CBC) substrate conformation, and (iii) the choice between squalene and 2,3-oxidosqualene as the substrate. We use these characters to construct four competing phylogenies to describe the evolution of polycyclic triterpenoid biosynthesis. The analysis suggests that malabaricanoids would be the most ancient polycyclic triterpenoids. The two most parsimonious evolutionary trees are the ones in which hopanoid and steroid cyclases diverged from a common ancestor. The transition from a CCC- to CBC-fold marks the major divergence in the evolution of these pathways, and it is diagnosable in the geological record. However, this transition does not require the simultaneous adoption of the aerobic substrate, 2,3-oxidosqualene, because these characters are controlled by independent parts of the enzyme. [source] ,Suicide' of crickets harbouring hairworms: a proteomics investigationINSECT MOLECULAR BIOLOGY, Issue 6 2006D. G. Biron Abstract Despite increasing evidence of host phenotypic manipulation by parasites, the underlying mechanisms causing infected hosts to act in ways that benefit the parasite remain enigmatic in most cases. Here, we used proteomics tools to identify the biochemical alterations that occur in the head of the cricket Nemobius sylvestris when it is driven to water by the hairworm Paragordius tricuspidatus. We characterized host and parasite proteomes during the expression of the water-seeking behaviour. We found that the parasite produces molecules from the Wnt family that may act directly on the development of the central nervous system (CNS). In the head of manipulated cricket, we found differential expression of proteins specifically linked to neurogenesis, circadian rhythm and neurotransmitter activities. We also detected proteins for which the function(s) are still unknown. This proteomics study on the biochemical pathways altered by hairworms has also allowed us to tackle questions of physiological and molecular convergence in the mechanism(s) causing the alteration of orthoptera behaviour. The two hairworm species produce effective molecules acting directly on the CNS of their orthoptera hosts. [source] H, feedback-control theory in biochemical systemsINTERNATIONAL JOURNAL OF ROBUST AND NONLINEAR CONTROL, Issue 1 2008E. Gershon Abstract In this paper we study the possible optimality of biochemical pathways in the H, sense. We start by presenting simple linearized models of single enzymatic reaction systems, where we apply classical and modern tools of feedback-control theory. We then apply the results obtained by our analysis to a linearly unbranched enzyme pathway system, where we explore the effect of a negative feedback loop internally exerted on the system by a self-product of the pathway. We then probe the sensitivity of the enzymatic system to variations in certain variables and we deal with the problem of assessing the optimality of the static-output feedback control, in the H, sense, inherent to the closed-loop system. In this point we demonstrate the applicability of our results via a theoretical example that provides an open-loop and closed-loop analysis of a four-block enzymatic system. We then apply the various tools we developed to the optimal analysis of the Threonine synthesis pathway which is regulated by three feedback loops. We demonstrate that this pathway is optimal in the H, sense, in the face of considerable uncertainties in the various enzyme concentrations of the pathway. Copyright © 2007 John Wiley & Sons, Ltd. [source] Microinjected neutrophils retain the ability to take up bacteriaJOURNAL OF ANATOMY, Issue 5 2002M. M. Bird It is now possible to microinject protein to probe specific biochemical pathways and/or cell functions in small cells such as human neutrophils (Bird et al. J.Anat.198, 2001). We have shown that these cells retain their ability to modify their F-actin cytoskeleton following the microinjection procedure. The principal task of neutrophils is to hunt and kill bacteria by responding to chemotactic gradients which cause them to extend actin rich pseudopodia in the direction of the highest concentration of these molecules. On reaching their target the neutrophils make tight contact with the bacteria and phagocytosis ensues. Here we address the question of whether or not the microinjected cells are still able to maintain their normal phagocytic activities. Human neutrophils maintained in culture for 20 mins were confronted with Staphylococcus aureus (1 × 104 cells/mL) for 5 min and then injected with rat IgG as an exogenous protein that also serves as a marker for injected cells. After 30 min the cells were fixed for fluorescence or confocal microscopy in 3.7% formaldehyde and permeabilised for 5 min (0.2% Triton X-100 in PBS). They were then incubated for 45 min in 2.5 µL FITC-anti rat IgG and 1 µL TRITC-phalloidin (to show the F-actin cytoskeleton), in 996.5 µL of PBS, washed 6 times in PBS and mounted on slides in 5 µL Mowiol containing a grain of antiquench. For TEM cells were fixed in 1.5% glutaraldehyde in cacodylate buffer for 3 min at room temperature and then washed in 0.2 m cacodylate buffer 6 times before incubation with 1 mm NiCl2 and SIGMA fast DAB peroxidase tablets for 30 min. The cells were postfixed in a 2% solution of osmium tetroxide for 30 min, dehydrated through a series of graded ethanols, and embedded and sectioned for TEM. By TEM the injected neutrophils were observed to have taken up bacteria into vacuoles of varying size. At the earliest stages of this process, prior to and immediately following the initial release of granular contents and the initiation of mechanisms to rapidly destroy bacteria, the bacteria fitted more tightly in the vacuoles than at later stages. Injected neutrophils commonly contained several bacteria; more than one bacterium was frequently located within a single vacuole of substantial size. Confocal laser microscopic observations confirmed that cells containing ingested bacteria also contained IgG. Thus injected cells not only survive the microinjection procedure but also retain their ability to take up bacteria and initiate the digestive process. [source] Mineral phosphate solubilization by rhizosphere bacteria and scope for manipulation of the direct oxidation pathway involving glucose dehydrogenaseJOURNAL OF APPLIED MICROBIOLOGY, Issue 1 2010B. Sashidhar Summary Microbial biodiversity in the soil plays a significant role in metabolism of complex molecules, helps in plant nutrition and offers countless new genes, biochemical pathways, antibiotics and other metabolites, useful molecules for agronomic productivity. Phosphorus being the second most important macro-nutrient required by the plants, next to nitrogen, its availability in soluble form in the soils is of great importance in agriculture. Microbes present in the soil employ different strategies to make use of unavailable forms of phosphate and in turn also help plants making phosphate available for plant use. Azotobacter, a free-living nitrogen fixer, is known to increase the fertility of the soil and in turn the productivity of different crops. The glucose dehydrogenase gene, the first enzyme in the direct oxidation pathway, contributes significantly to mineral phosphate solubilization ability in several Gram-negative bacteria. It is possible to enhance further the biofertilizer potential of plant growth-promoting rhizobacteria by introducing the genes involved mineral phosphate solubilization without affecting their ability to fix nitrogen or produce phytohormones for dual benefit to agricultural crops. Glucose dehydrogenases from Gram-negative bacteria can be engineered to improve their ability to use different substrates, function at higher temperatures and EDTA tolerance, etc., through site-directed mutagenesis. [source] Metabolomics in the assessment of chemical-induced reproductive and developmental outcomes using non-invasive biological fluids: application to the study of butylbenzyl phthalateJOURNAL OF APPLIED TOXICOLOGY, Issue 8 2009Susan Sumner Abstract This study was conducted to evaluate the use of metabolomics for improving our ability to draw correlations between early life exposures and reproductive and/or developmental outcomes. Pregnant CD rats were exposed by gavage daily during gestation to vehicle or to butylbenzyl phthalate (BBP) in vehicle at a level known to induce effects in the offspring and at a level previously not shown to induce effects. Urine was collected for 24,h (on dry ice using all glass metabolism chambers) from dams on gestational day 18 (during exposure) and on post natal day (pnd) 21, and from pnd 25 pups. Traditional phenotypic anchors were measured in pups (between pnd 0 and pnd 26). Metabolomics of urine collected from dams exposed to vehicle or BBP exhibited different patterns for endogenous metabolites. Even three weeks after gestational exposure, metabolic profiles of endogenous compounds in urine could differentiate dams that received the vehicle, low dose or high dose of BBP. Metabolic profiles could differentiate male from female pups, pups born to dams receiving the vehicle, low or high BBP dose, and pups with observable adverse reproductive effects from pups with no observed effects. Metabolites significant to the separation of dose groups and their relationship with effects measured in the study were mapped to biochemical pathways for determining mechanistic relevance. The application of metabolomics to understanding the mechanistic link between low levels of environmental exposure and disease/dysfunction holds huge promise, because this technology is ideal for the analysis of biological fluids in human populations. Copyright © 2009 John Wiley & Sons, Ltd. [source] The roles of calcium/calmodulin-dependent and Ras/mitogen-activated protein kinases in the development of psychostimulant-induced behavioral sensitizationJOURNAL OF NEUROCHEMISTRY, Issue 1 2003Stephanie C. Licata Abstract Although the development of behavioral sensitization to psychostimulants such as cocaine and amphetamine is confined mainly to one nucleus in the brain, the ventral tegmental area (VTA), this process is nonetheless complex, involving a complicated interplay between neurotransmitters, neuropeptides and trophic factors. In the present review we present the hypothesis that calcium-stimulated second messengers, including the calcium/calmodulin-dependent protein kinases and the Ras/mitogen-activated protein kinases, represent the major biochemical pathways whereby converging extracellular signals are integrated and amplified, resulting in the biochemical and molecular changes in dopaminergic neurons in the VTA that represent the critical neuronal correlates of the development of behavioral sensitization to psychostimulants. Moreover, given the important role of calcium-stimulated second messengers in the expression of behavioral sensitization, these signal transduction systems may represent the biochemical substrate through which the transient neurochemical changes associated with the development of behavioral sensitization are translated into the persistent neurochemical, biochemical and molecular alterations in neuronal function that underlie the long-term expression of psychostimulant-induced behavioral sensitization. [source] Differential apoptotic response of J774 macrophages to alumina and ultra-high-molecular-weight polyethylene particlesJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 1 2002Alain Petit We recently identified apoptosis in in vitro wear particle-stimulated macrophages. The recent explosion of interest in apoptosis lies in the fact that it is under positive and negative regulation through evolutionary conserved biochemical pathways. It may also be possible to modulate macrophage apoptosis in the treatment of periprosthetic osteolysis. The purpose of this study was to compare the macrophage response to identically sized particles of alumina ceramic (Al2O3) and ultra-high-molecular-weight polyethylene (UHMWPE) in terms of TNF-, release and induction of apoptosis. J774 mouse macrophages were incubated for 0,24 h in the presence of Al2O3 and UHMWPE particles. TNF-, release was measured by ELISA; Poly(ADP-ribose)polymerase (PARP) and caspase-3 expression was analyzed by Western blot; DNA fragmentation (DNA laddering) was visualized on agarose gel containing ethidium bromide. Al2O3 particles induced TNF-, release after 4 h incubation with concentrations reaching 483 and 800 pg/ml after 24 h with 125 and 250 particles/macrophage, respectively (control = 161 pg/ml) (P < 0.05 vs. control). The same concentrations of UHMWPE particles induced a much larger and significant TNF-, release after only 1 h incubation, increasing up to 6250 pg/ml after 24 h (P < 0.05 vs. control). Western blot analysis demonstrated that the active caspase-3 fragment (17 kDa) and the proteolytic PARP fragment (85 kDa) were expressed after 2 h incubation with 125 and 250 Al2O3 particles/macrophage. The active caspase-3 and the PARP fragment had lower expression and appeared after a longer incubation time (8 h) with 125 and 250 UHMWPE particles/macrophage. Finally, DNA fragmentation (DNA laddering) was observed after 16 h with 125 and 250 particles of Al2O3 per macrophage whereas no laddering was induced by UHMWPE particles even after 24 h incubation. This study shows that although both Al2O3 and UHMWPE particles induce TNF-, release, this stimulation was much greater (8,10 times higher) with UHMWPE than A12O3 (P < 0.05 vs. control). As well, the induction of apoptosis, as measured by activation of caspase-3, PARP cleavage and DNA laddering, is different for these two particles, being faster and more important with Al2O3 than UHMWPE. We hypothesize that the ability of Al2O3 to induce macrophage apoptosis may explain the lower TNF-, release observed with these particles and explain the differences seen in osteolysis patterns of ceramic,ceramic (CC) vs. metal,polyethylene (Mpe) articulations. In conclusion, apoptosis may be a major internal mechanism to decrease macrophage activity and may be a desired therapeutic endpoint. The identification of an apoptosis-related pathway in the macrophage response to ceramic particles provides crucial data for a rational approach in the treatment and/or prevention of periprosthetic osteolysis. © 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved. [source] Ischemic preconditioning attenuates the oxidant-dependent mechanisms of reperfusion cell damage and death in rat liverLIVER TRANSPLANTATION, Issue 11 2002Barbara Cavalieri In an in vivo rat model of liver ischemia followed by reperfusion a consistent appearance of necrosis and activation of biochemical pathways of apoptosis was reproduced and monitored after 30 minutes reperfusion. Preconditioning by application of a short cycle of ischemia-reperfusion (10 minutes + 10 minutes) positively conditioned recovery of the organ at reperfusion, attenuating both necrotic and apoptotic events. Preconditioning at least halved cell oxidative damage occurring early at reperfusion, and as a major consequence, the increase of cytolysis and apoptosis occurring at reperfusion was about 50% less. The attenuation of both pathways of cell death by preconditioning appeared at least partly related to its modulate action on H2O2 and 4-hydroxy-2,3-trans-nonenal production. The overall data point to a marked diminished oxidant generation and oxidative reactions as one major possible mechanism through which ischemic preconditioning exerts protection against necrotic and apoptotic insult to the postischemic liver. [source] Three temporal classes of gene expression during the Chlamydia trachomatis developmental cycleMOLECULAR MICROBIOLOGY, Issue 4 2000E. I. Shaw The obligate intracellular bacterium Chlamydia trachomatis has a unique developmental cycle that involves functionally and morphologically distinct cell types adapted for extracellular survival and intracellular multiplication. Infection is initiated by an environmentally resistant cell type called an elementary body (EB). Over the first several hours of infection, EBs differentiate into a larger replicative form, termed the reticulate body (RB). Late in the infectious process, RBs asynchronously begin to differentiate back to EBs, which accumulate within the lumen of the inclusion until released from the host cell for subsequent rounds of infection. In an effort to characterize temporal gene expression in relation to the chlamydial developmental cycle, we have used quantitative,competitive polymerase chain reaction (QC-PCR) and reverse transcription (RT)-PCR techniques. These analyses demonstrate that C. trachomatis double their DNA content every 2,3 h, with synthesis beginning between 2 and 4 h after infection. We determined the onset of transcription of specific temporal classes of developmentally expressed genes. RT-PCR analysis was performed on several genes encoding key enzymes or components of essential biochemical pathways and functions. This comparison encompassed approximately 8% of open reading frames on the C. trachomatis genome. In analysis of total RNA samples harvested at 2, 6, 12 and 20 h after infection, using conditions under which a single chlamydial transcript per infected cell is detected, three major temporal classes of gene expression were resolved. Initiation of transcription appears to occur in three temporal classes which we have operationally defined as: early, which are detected by 2 h after infection during the germination of EBs to RBs; mid-cycle, which appear between 6 and 12 h after infection and represent transcripts expressed during the growth and multiplication of RBs; or late, which appear between 12 and 20 h after infection and represent those genes transcribed during the terminal differentiation of RBs to EBs. Collectively, the data suggest that chlamydial early gene functions are weighted toward initiation of macromolecular synthesis and the establishment of their intracellular niche by modification of the inclusion membrane. Surprisingly, representative enzymes of intermediary metabolism and structural proteins do not appear to be transcribed until 10,12 h after infection; coinciding with the onset of observed binary fission of RBs. Late gene functions appear to be predominately those associated with the terminal differentiation of RBs back to EBs. [source] Effects of etomidate on free intracellular amino acid concentrations in polymorphonuclear leucocytes in vitroACTA ANAESTHESIOLOGICA SCANDINAVICA, Issue 4 2000J. Mühling Background: Previous studies have shown the inhibitory effects of etomidate on polymorphonuclear leucocyte (PMN) function. No reports exist, however, regarding free intracellular amino acid metabolism, although physiological cell metabolism and basic cell functions rely upon a balanced intracellular amino acid content and the cell membrane-mediated separation of cellular amino acids from the extracellular plasma amino acid pool. Thus, in the current study, we evaluated the effects of etomidate on free intracellular amino acid metabolism in PMN. Methods: With ethics committee approval, blood was withdrawn from 35 healthy volunteers and incubated (1 h) either with 0 ,g/ml, 0.0156 ,g/ml, 0.0625 ,g/ml or 0.5 ,g/ml of etomidate as well as with its additives (propylene glycol and Lipofundin MCT® 10%). The PMN were separated using standardized Percoll® -gradient and centrifugation procedure before deep-freezing and lyophilization techniques were employed. All PMN samples were dissolved in methanol/H2O, and the concentrations of free intracellular amino acids were monitored using both novel advanced PMN-separation and high-performance liquid chromatography techniques. Results: Etomidate influenced important free amino acid profiles in PMN in a dose-dependent manner, indicating complex changes of cellular amino acid turnover. Neither propylene glycol nor Lipofundin MCT® 10% changed free amino acid concentrations in PMN. Conclusions: For the first time, the effects of etomidate on free intracellular amino acid metabolism in PMN have been investigated. Our results draw attention to the biochemical pathways which may be involved in etomidate-induced alterations in PMN function and cellular immunocompetence. [source] Variation in oxygen isotope fractionation during cellulose synthesis: intramolecular and biosynthetic effectsPLANT CELL & ENVIRONMENT, Issue 10 2006LEONEL STERNBERG ABSTRACT The oxygen isotopic composition of plant cellulose is commonly used for the interpretations of climate, ecophysiology and dendrochronology in both modern and palaeoenvironments. Further applications of this analytical tool depends on our in-depth knowledge of the isotopic fractionations associated with the biochemical pathways leading to cellulose. Here, we test two important assumptions regarding isotopic effects resulting from the location of oxygen in the carbohydrate moiety and the biosynthetic pathway towards cellulose synthesis. We show that the oxygen isotopic fractionation of the oxygen attached to carbon 2 of the glucose moieties differs from the average fractionation of the oxygens attached to carbons 3,6 from cellulose by at least 9%, for cellulose synthesized within seedlings of two different species (Triticum aestivum L. and Ricinus communis L.). The fractionation for a given oxygen in cellulose synthesized by the Triticum seedlings, which have starch as their primary carbon source, is different than the corresponding fractionation in Ricinus seedlings, within which lipids are the primary carbon source. This observation shows that the biosynthetic pathway towards cellulose affects oxygen isotope partitioning, a fact heretofore undemonstrated. Our findings may explain the species-dependent variability in the overall oxygen isotope fractionation during cellulose synthesis, and may provide much-needed insight for palaeoclimate reconstruction using fossil cellulose. [source] Proteomics: New insights into rheumatic diseasesPROTEOMICS - CLINICAL APPLICATIONS, Issue 2 2009Emilio Camafeita Abstract Tremendous advances undergone in electrophoresis, chromatography, and MS have led proteomic research to unprecedented achievement over the last decade. Proteomics is presently employed for assessing protein expression levels, for monitoring cellular activities and for determination of biochemical pathways, revolutionizing the way we study disease by opening up the possibility to decipher the pathogenesis of clinical manifestations. Over 200 disorders including osteoarthritis (OA), rheumatoid arthritis (RA), and osteoporosis are considered rheumatic diseases (RDs), which affect the musculoskeletal system (joints and other supporting structures of the body such as muscles, tendons, ligaments, and bones) and are a leading cause of disability among older adults. Despite that an autoimmune origin has been proposed for some RDs like RA, the pathogenesis of most of these diseases is still unclear. Therefore, proteomic research on RDs, notably OA and RA, can help clarify underlying disease mechanisms, develop biomarkers to improve early detection, measure response to treatment, and devise new therapies. Achievements in the field of proteomics research on RDs are summarized in this work. [source] Neuroproteomics and its applications in research on nicotine and other drugs of abusePROTEOMICS - CLINICAL APPLICATIONS, Issue 11 2007Ming D. Li Dr. Abstract The rapidly growing field of neuroproteomics is able to track changes in protein expression and protein modifications underlying various physiological conditions, including the neural diseases related to drug addiction. Thus, it presents great promise in characterizing protein function, biochemical pathways, and networks to understand the mechanisms underlying drug dependence. In this article, we first provide an overview of proteomics technologies and bioinformatics tools available to analyze proteomics data. Then we summarize the recent applications of proteomics to profile the protein expression pattern in animal or human brain tissues after the administration of nicotine, alcohol, amphetamine, butorphanol, cocaine, and morphine. By comparing the protein expression profiles in response to chronic nicotine exposure with those appearing in response to treatment with other drugs of abuse, we identified three biological processes that appears to be regulated by multiple drugs of abuse: energy metabolism, oxidative stress response, and protein degradation and modification. Such similarity indicates that despite the obvious differences among their chemical properties and the receptors with which they interact, different substances of abuse may cause some similar changes in cellular activities and biological processes in neurons. [source] Function of plastidial pyruvate kinases in seeds of Arabidopsis thaliana,THE PLANT JOURNAL, Issue 3 2007Sébastien Baud Summary Pyruvate kinase (PK) catalyses the irreversible synthesis of pyruvate and ATP, which are both used in multiple biochemical pathways. These compounds are essential for sustained fatty acid production in the plastids of maturing Arabidopsis embryos. Using a real-time quantitative reverse transcriptase (RT)-PCR approach, the three genes encoding putative plastidial PKs (PKps) in Arabidopsis, namely PKp1 (At3g22960), PKp2 (At5g52920) and PKp3 (At1g32440), were shown to be ubiquitously expressed. However, only PKp1 and PKp2 exhibited significant expression in maturing seeds. The activity of PKp1 and PKp2 promoters was consistent with this pattern, and the study of the PKp1:GFP and PKp2:GFP fusion proteins confirmed the plastidial localization of these enzymes. To further investigate the function of these two PKp isoforms in seeds comprehensive functional analyses were carried out, including the cytological, biochemical and molecular characterization of two pkp1 and two pkp2 alleles, together with a pkp1pkp2 double mutant. The results obtained outlined the importance of these PKps for fatty acid synthesis and embryo development. Mutant seeds were depleted of oil, their fatty acid content was drastically modified, embryo elongation was retarded and, finally, seed germination was also affected. Together, these results provide interesting insights concerning the carbon fluxes leading to oil synthesis in maturing Arabidopsis seeds. The regulation of this metabolic network by the WRINKLED1 transcription factor is discussed, and emphasizes the role of plastidial metabolism and the importance of its tight regulation. [source] | |||||||||||||||||||||||||||||||||||||