Analysis of biotin biosynthesis pathway in coryneform bacteria: Brevibacterium flavum.
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Biotin↗
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Results for “Pathway analysis”
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A hitchhiker's guide to analysis of the secretory pathway in yeast.
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Biological Transport↗
Pregnenolone metabolized to 17alpha-hydroxyprogesterone in yeast: biochemical analysis of a metabolic pathway.
The cDNA coding for the human 3beta-hydroxy-5-ene steroid dehydrogenase/5-ene-4-ene steroid isomerase (3beta-HSD) has been expressed in yeast. When expressed from identical vectors except for the coding sequence, the specific activity of the type I is lower than that of the type II enzyme. A mutant of the human 3beta-HSD type II lacking the putative membrane spanning domain 1 was generated by site directed mutagenesis: its apparent K(m) for pregnenolone (PREG) is significantly increased and its V reduced to the level of the type I enzyme. The influence of the kinetic properties of 3beta-HSD in the accumulation of 17alpha-hydroxyprogesterone was probed by co-expression of the bovine 17alpha-hydroxylase cytochrome P450 (P45017alpha) cDNA. The metabolism of PREG was followed with time using the membrane fraction. Kinetic properties of the 3beta-HSD were modulated such that its activity was in excess, limiting or balanced with respect to the activity of the P45017alpha and the accumulation of intermediates and products recorded. Conditions for the generation of the by-products resulting from the 17,20-Lyase activity of the P45017alpha were found. The potential applications of the system are discussed.
17-alpha-Hydroxyprogesterone↗
Analysis of Dishevelled signalling pathways during Xenopus development.
BACKGROUND: Recent studies have demonstrated that the Wnt, Frizzled and Notch proteins are involved in a variety of developmental processes in fly, worm, frog and mouse embryos. The Dishevelled (Dsh) protein is required for Drosophila cells to respond to Wingless, Notch and Frizzled signals, but the molecular mechanisms of its action are not well understood. Using the ability of a mutant form of the Xenopus homologue of Dsh (Xdsh) to block Wnt and Dsh signalling in a model system, this work attempts to clarify the role of the endogenous Xdsh during the early stages of vertebrate development. RESULTS: A mutant Xdsh (Xdd1) with an internal deletion of the conserved PDZ/DHR domain was constructed. Overexpression of Xdd1 mRNA in ventral blastomeres of Xenopus embryos strongly inhibited induction of secondary axes by the wild-type Xdsh and Xwnt8 mRNAs, but did not affect the axis-inducing ability of beta-catenin mRNA. These observations suggest that Xdd1 acts as a dominant-negative mutant. Dorsal expression of Xdd1 caused severe posterior truncations in the injected embryos, whereas wild-type Xdsh suppressed this phenotype. Xdd1 blocked convergent extension movements in ectodermal explants stimulated with mesoderm-inducing factors and in dorsal marginal zone explants, but did not affect mesoderm induction and differentiation. CONCLUSIONS: A vertebrate homologue of Dsh is a necessary component of Wnt signal transduction and functions upstream of beta-catenin. These findings also establish a requirement for the PDZ domain in signal transduction by Xdsh, and suggest that endogenous Xdsh controls morphogenetic movements in the embryo.
Adaptor Proteins, Signal Transducing↗
Analysis of dibenzothiophene metabolic pathway in Mycobacterium strain G3.
The dibenzothiophene (DBT) metabolic pathway in Mycobacterium strain G3, which is classified as a desulfurizing microorganism with the 4S pathway, was analyzed. 2-Hydroxybiphenyl (HBP), which is an end metabolite in the DBT desulfurization reaction, and 2-methoxybiphenyl (MBP) were found in the reaction mixture, and the methoxylation pathway from HBP to MBP was clarified. Although the substrate in the methoxylation reaction was HBP, there was no relationship between expression of the methoxylation activity and that of the desulfurization activity. Then, 4,6-dimethyl DBT, 4,6-diethyl DBT and benzo[b]naphtho[2,1-d]thiophene were metabolized to their methoxy forms via the desulfurization pathway. We established the methoxylation pathway in Mycobacterium G3.
Journal Article↗
Pharmacological analysis of signal transduction pathways required for oxidative burst in chicken heterophils stimulated by a Toll-like receptor 2 agonist.
Toll-like receptors (TLRs) play an important role in the innate immune response of avian heterophils. We previously used the pharmacological inhibitors genistein, verapamil, chelerythrine, and pertussis toxin to investigate the upstream signaling events involved in TLR2-mediated oxidative burst in chicken heterophils. Only chelerythrine, a protein kinase C inhibitor, was found to significantly inhibit oxidative burst stimulated by the TLR2 agonist lipoteichoic acid (LTA). In the present study, we used selective pharmacological inhibitors to investigate the roles of phosphatidylinositol-3'-kinase (PI3-K), phospholipase C (PLC), calcium-dependent protein kinase C (PKC), extra-cellular signal regulated kinase (ERK), and nuclear translocation factor kappa B (NF-kappaB) on TLR2-mediated oxidative burst. U-73122 (a PLC inhibitor), wortmannin (a PI3-K inhibitor), PD 98059 (an ERK inhibitor), Gö 6976 (a PKC inhibitor) and Bay 11-7082 (a NF-kappaB inhibitor) significantly decreased LTA-stimulated oxidative burst in heterophils by 77%, 30%, 36%, 78%, and 61%, respectively. Activated TLR2 utilizes PI3-K, PLC, PKC, ERK, and NF-kappaB as signaling factors that mediate the oxidative burst of chicken heterophils.
Androstadienes↗
Computer simulation and analysis of the reaction pathway of triosephosphate isomerase.
A theoretical approach designed for chemical reactions in the condensed phase is used to determine the energy along the reaction path of the enzyme triosephosphate isomerase. The calculations address the role of the enzyme in lowering the barrier to reaction and provide a decomposition into specific residue contributions. The results suggest that, although Lys-12 is most important, many other residues within 16 A of the substrate contribute and that histidine-95 as the imidazole/imidazolate pair could act as an acid/base catalyst.
Amino Acid Sequence↗
Mechanism of aminoacylation of transfer RNA. A pre-steady-state analysis of the reaction pathway catalyzed by the methionyl-tRNA synthetase of Bacillus stearothermophilus.
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Amino Acyl-tRNA Synthetases↗
Effects of mutations in plastocyanin on the kinetics of the protein rearrangement gating the electron-transfer reaction with zinc cytochrome c. Analysis of the rearrangement pathway.
We study, by flash kinetic spectrophotometry on the microsecond time scale, the effects of ionic strength and viscosity on the kinetics of oxidative quenching of the triplet state of zinc cytochrome c (3Zncyt) by the wild-type form and the following nine mutants of cupriplastocyanin: Leu12Glu, Leu12Asn, Phe35Tyr, Gln88Glu, Tyr83Phe, Tyr83His, Asp42Asn, Glu43Asn, and the double mutant Glu59Lys/Glu60Gln. The unimolecular rate constants for the quenching reactions within the persistent diprotein complex, which predominates at low ionic strengths, and within the transient diprotein complex, which is involved at higher ionic strengths, are equal irrespective of the mutation. Evidently, the two complexes are the same. In both reactions, the rate-limiting step is rearrangement of the diprotein complex from a configuration optimal for docking to the one optimal for the subsequent electron-transfer step, which is fast. We investigate the effects of plastocyanin mutations on this rearrangement, which gates the overall electron-transfer reaction. Conversion of the carboxylate anions into amide groups in the lower acidic cluster (residues 42 and 43), replacement of Tyr83 with other aromatic residues, and mutations in the hydrophobic patch in plastocyanin do not significantly affect the rearrangement. Conversion of a pair of carboxylate anions into a cationic and a neutral residue in the upper acidic cluster (residues 59 and 60) impedes the rearrangement. Creation of an anion at position 88, between the upper acidic cluster and the hydrophobic patch, facilitates the rearrangement. The rate constant for the rearrangement smoothly decreases as the solution viscosity increases, irrespective of the mutation. Fittings of this dependence to the modified Kramers's equation and to an empirical equation show that zinc cytochrome c follows the same trajectory on the surfaces of all the plastocyanin mutants but that the obstacles along the way vary as mutations alter the electrostatic potential. Mutations that affect protein association (i.e., change the binding constant) do not necessarily affect the reaction between the associated proteins (i.e., the rate constant) and vice versa. All of the kinetic and thermodynamic effects and noneffects of mutations consistently indicate that in the protein rearrangement the basic patch of zinc cytochrome c moves from a position between the two acidic clusters to a position at or near the upper acidic cluster.
Animals↗
Functional proteomics analysis of signal transduction pathways of the platelet-derived growth factor beta receptor.
We report efficient methods for using functional proteomics to study signal transduction pathways in mouse fibroblasts following stimulation with PDGF. After stimulation, complete cellular proteins were separated using two-dimensional electrophoresis and phosphorylated proteins were detected with anti-phosphotyrosine and anti-phosphoserine antibodies. About 260 and 300 phosphorylated proteins were detected with the anti-phosphotyrosine and anti-phosphoserine antibodies, respectively, at least 100 of which showed prominent changes in phosphorylation as a function of time after stimulation. Proteins showing major time-dependent changes in phosphorylation were subjected to in-gel digestion with trypsin and identified by mass spectroscopy using MALDI-TOF mass fingerprinting and ESI peptide sequencing. We have observed phosphorylated proteins known to be part of the PDGF signal transduction pathway such as ERK 1, serine/threonine protein kinase akt and protein tyrosine phosphatase syp, proteins such as proto-oncogene tyrosine kinase fgr previously known to participate in other signal transduction pathways, and some proteins such as plexin-like protein with no previously known function in signal transduction. Information about the phosphorylation site was obtained for proto-oncogene tyrosine kinase fgr and for cardiac alpha-actin. The methods used here have proven to be suitable for the identification of time-dependent changes in large numbers of proteins involved in signal transduction pathways.
3T3 Cells↗
Density functional theory analysis of the reaction pathway for methane oxidation to acetic acid catalyzed by Pd2+ in sulfuric acid.
Density functional theory has been used to investigate the thermodynamics and activation barriers associated with the direct oxidation of methane to acetic acid catalyzed by Pd2+ cation in concentrated sulfuric acid. Pd2+ cations in such solutions are ligated by two bisulfate anions and by one or two molecules of sulfuric acid. Methane oxidation is initiated by the addition of CH4 across one of the Pd-O bonds of a bisulfate ligand to form Pd(HSO4)(CH3)(H2SO4)2. The latter species will react with CO to produce Pd(HSO4)(CH3CO)(H2SO4)2. The most likely path to the final products is found to be via oxidation of Pd(HSO4)(CH3)(H2SO4)2 and Pd(HSO4)(CH3CO)(H2SO4)2 to form Pd(eta2-HSO4)(HSO4)2(CH3)(H2SO4) and Pd(eta2-HSO4)(HSO4)2(CH3CO)(H2SO4), respectively. CH3HSO4 or CH3COHSO4 is then produced by reductive elimination from the latter two species, and CH(3)COOH is then formed by hydrolysis of CH3COHSO4. The loss of Pd2+ from solution to form Pd(0) or Pd-black is predicted to occur via reduction with CO. This process is offset, though, by reoxidation of palladium by either H2SO4 or O2.
Journal Article↗
Biosynthetic pathway and gene cluster analysis of curacin A, an antitubulin natural product from the tropical marine cyanobacterium Lyngbya majuscula.
Curacin A (1) is a potent cancer cell toxin obtained from strains of the tropical marine cyanobacterium Lyngbya majuscula found in Curaçao. Its structure is unique in that it contains the sequential positioning of a thiazoline and cyclopropyl ring, and it exerts its potent cell toxicity through interaction with the colchicine drug binding site on microtubules. A series of stable isotope-labeled precursors were fed to cultures of curacin A-producing strains and, following NMR analysis, allowed determination of the metabolic origin of all atoms in the natural product (one cysteine, 10 acetate units, two S-adenosyl methionine-derived methyl groups) as well as several unique mechanistic insights. Moreover, these incorporation experiments facilitated an effective gene cloning strategy that allowed identification and sequencing of the approximately 64 kb putative curacin A gene cluster. The metabolic system is comprised of a nonribosomal peptide synthetase (NRPS) and multiple polyketide synthases (PKSs) and shows a very high level of collinearity between genes in the cluster and the predicted biochemical steps required for curacin biosynthesis. Unique features of the cluster include (1) all but one of the PKSs are monomodular multifunctional proteins, (2) a unique gene cassette that contains an HMG-CoA synthase likely responsible for formation of the cyclopropyl ring, and (3) a terminating motif that is predicted to function in both product release and terminal dehydrative decarboxylation.
Amino Acid Sequence↗
Theoretical analysis of the reaction pathway and the effect of the axial ligand for 3-oxobutylideneaminatocobalt(II)-catalyzed cyclopropanation.
[structure: see text] The reaction pathway of the cyclopropanation catalyzed by the 3-oxobutylideneaminatocobalt(II) complex was analyzed by the density functional method to reveal that the axial donor ligand produced two prominent effects. One is that the activation energy for the formation of the cobalt carbene complex was reduced and that the activation energy for the cyclopropanation step was increased. The other is that the distance of the carbene carbon above the ligand plane was shortened during the cyclopropanation step.
Journal Article↗
From patterns to pathways: gene expression data analysis comes of age.
Many different biological questions are routinely studied using transcriptional profiling on microarrays. A wide range of approaches are available for gleaning insights from the data obtained from such experiments. The appropriate choice of data-analysis technique depends both on the data and on the goals of the experiment. This review summarizes some of the common themes in microarray data analysis, including detection of differential expression, clustering, and predicting sample characteristics. Several approaches to each problem, and their relative merits, are discussed and key areas for additional research highlighted.
Cluster Analysis↗
The diverse roles of ubiquitin and the 26S proteasome in the life of plants.
A tightly regulated and highly specific system for the degradation of individual proteins is essential for the survival of all organisms. In eukaryotes, this is achieved by the tagging of proteins with ubiquitin and their subsequent recognition and degradation by the 26S proteasome. In plants, genetic analysis has identified many genes that regulate developmental pathways. Subsequent analysis of these genes has implicated ubiquitin and the 26S proteasome in the control of diverse developmental processes, and indicates that proteolysis is a crucial regulatory step throughout the life cycle of plants.
Hydrolysis↗
Analysis of fluid transport pathways and their determinants in peritoneal dialysis patients with ultrafiltration failure.
Ultrafiltration failure (UFF) is a serious complication of peritoneal dialysis (PD). The aim of the study was to analyze changes in water transport and their determinants in UFF patients over the time on PD. Standard peritoneal permeability analyses of 50 stable PD patients with UFF were analyzed. Fluid transport through small pores (SPT), free water transport (FWT) at 60 min, their contributions on total ultrafiltration (SPTC and FWTC), and their determinants were assessed. Patients were divided in Group I (UFF) treated for less than 24 months, Group II treated 24-60 months, and Group III treated for more than 60 months. Group I (UFF) was compared with Group I (non-UFF) matched for the duration of PD treatment and age. Transcapillary ultrafiltration (TCUF), SPT, FWT, and FWTC were significantly lower in Group III when compared to the other UFF groups. In this group also, negative relationship was present between FWT, the ultrafiltration coefficient LpA, and osmotic conductance to glucose on one hand and PD duration on the other. FWT was positively related to osmotic conductance to glucose in all groups. Group I (UFF) showed significantly higher solute transport, effective lymphatic absorption rate, lower TCUF, and lower FWT than Group I (non-UFF). The patterns of UFF in PD patients are dependent on the duration of treatment.
Adult↗
Application of electrical analogues for control analysis of simple metabolic pathways.
I have used electrical analogues for calculating the Flux Control Coefficients of metabolic pathways. An analogue circuit consists of resistances that are connected in series (or parallel) with a voltage (or current) source. In constructing the analogues, each of the enzymes in the pathway is associated with a resistance whose magnitude depends on the Elasticity Coefficients of the enzymes. These circuits can be designed in a heuristic fashion directly from the configuration of the pathway, without the necessity of writing down the governing equations with the use of Summation and Connectivity Theorems. The Flux Control Coefficients of the enzymes are represented by voltages across (or currents through) the resistances and are determined by an application of Ohm's Law. Results are given for (a) a simple linear pathway without feedback or feedforward regulation, and (b) a linear pathway with feedback inhibition. The analogue circuits are also convenient for assessing the relative importance of the various enzymes in flux control, and for simplifying the structure of a given pathway.
Electricity↗
Genetic analysis of the signalling pathway activated by external amino acids in Saccharomyces cerevisiae.
The permease-like amino acid sensor Ssy1p of Saccharomyces cerevisiae is required for transcriptional induction, in response to external amino acids, of several genes encoding peptide and amino acid permeases. Among them is AGP1 encoding a low-affinity, broad-specificity amino acid permease important for the utilization of amino acids as a nitrogen source. We report here data from experiments aimed at identifying components of the signalling pathway activated by Ssy1p. Overproduction of the large amino-terminal tail of Ssy1p interferes negatively with the induction of AGP1 in wild-type cells. Furthermore, overproduction of this domain can relieve growth defects of a ssy1 null strain, indicating that the N-terminal tail of Ssy1p is an important functional element of the pathway. Consistent with a role for Ssy1p in the recognition of amino acids, a mutant form of the protein with a Thr to Ile substitution in the eighth predicted transmembrane domain is competent for the induction of AGP1 by leucine but not by other amino acids. In a screen for other mutants defective in the Ssy1p pathway, we confirmed that PTR3 and SSY5 encode additional factors essential for AGP1 expression in response to multiple amino acids. Data obtained by overproducing Ptr3p and Ssy5p in ssy1Delta, ptr3Delta and ssy5Delta mutants suggest that Ptr3p acts downstream from Ssy1p and Ssy5p downstream from Ptr3p in the transduction pathway. Furthermore, two-hybrid experiments indicated that Ptr3p interacts with Ssy5p and that Ptr3p can self-associate. Finally, the Cys-6-Zn2 transcription factor Uga35p/Dal81p required for the induction of AGP1 is also essential for the expression of two other genes under Ssy1p-Ptr3p-Ssy5p control, namely BAP2 and PTR2, suggesting that the protein is yet another component of the amino acid signalling pathway.
Amino Acid Transport Systems, Neutral↗