Two-step Activation Research Articles

Two-step activation of valproic acid (VPA) by hepatic cytochrome P-450

There is now considerable evidence that the unsaturated metabolite of VPA, Δ 4-VPA, may be responsible for VPA-induced hepatic injury. It is proposed that VPA is activated to a hepatotoxic species by a two-step cytochrome P-450-mediated mechanism consisting of 1) desaturation and 2) unknown activation, possibly by epoxide formation, involving one or more P-450 isozymes with subsequent covalent binding to cellular macromolecules. In vivo and in vitro experimental protocols are indicated for testing the proposed hypothesis.

Expression of lupin nodulin genes during root nodule development

Seven lupin cDNA clones were used to study the expression of corresponding genes during nodule development by Northern blots analysis. They include six nodulin cDNAs: pLLb (lupin leghemoglobin), pLN 13, pLN 21–27, pLN 281, pLN 50, pLNGS (nodule form of glutamine synthetase GS n and root form of GS: pGS. The appearance of nodulin mRNAs during lupin nodule development showed that the nodulin sequences analysed represent a group of plant genes involved in the nitrogen fixation process rather than formation of nodule. This is based on the observation that they are activated at the time when the nodule has already been formed, prior to the onset of nitrogenase activity. The products of Lb, nodulin 21–67, the nodulin coded by pLN13 and the nodulin 281 genes appeared between 11 and 13 days after infection, whereas the nodulion coded by pLN50 and the nodule form of GS appeared 18 days after inoculation. Twenty-one days post-infection a dramatic increase in the transciption rates of all nodulin genes is observed. This phenomenon may be related to the onset of nitrogenase activity. The possible mechanism of two-step activation of nodulin genes is discussed.

Thyroid hormone induction of keratin genes: a two-step activation of gene expression during development.

To determine the mechanism of action of the thyroid hormone triiodothyronine (T3) during metamorphosis of the amphibian epidermis, we have investigated the developmental activation of the 63-kD keratin genes in the frog Xenopus laevis. These genes code for three closely related keratins that first appear in the larval epidermis and accumulate during metamorphosis to become the most abundant proteins in the adult epidermis. We report here that the 63-kD keratins and their mRNAs first appear at stages 48-52. The level of 63-kD keratin gene expression remains relatively low until stage 56 and then increases dramatically. Quantitative analysis of the concentration of 63-kD keratin mRNA demonstrates low levels until stage 55/56, followed by an increase greater than 300-fold from stage 55/56 to the adult. Each adult cell contains approximately 55,000 molecules of the 63-kD keratin mRNAs. T3 is not required for the initial activation of the 63-kD keratin genes, but high-level expression is absolutely dependent on T3. High-level expression is prevented by propylthiouracil, which inhibits thyroid hormone synthesis and can be induced precociously both in vivo and in vitro with exogenous T3 as early as stage 48, but not prior to that time. Thus, the full activation of the 63-kD keratin genes during development requires two regulatory steps, one independent and one dependent on T3.

Pathobiochemical mechanisms of hepatocellular damage following lipid peroxidation

In hepatocytes, cytotoxic events induced by haloalkanes or acute iron-overload exhibit neither a quantitative nor a temporal correlation to lipid peroxidation or covalent binding. Thus, secondary pathological mechanisms have been postulated linking initial focal reactions of free radicals and end stage pathological consequences. Due to the crucial role of plasma-membrane integrity in the cytotoxic process it has to be supposed that relevant secondary pathological mechanisms finally impair the physico-chemical and functional properties of this membrane. Based on recent developments a chain of causality is proposed as a two-step activation of phospholipase A 2 producing cytolytic amounts of lysophosphatides. In this cascade, the initial activating step is a decrease of membrane lipid fluidity induced by lipid peroxidation and/or by calcium binding and intramembranous formation of 4-hydroxynonenal. This enzyme activation is further amplified by the early rise of cytosolic calcium. Consequently, increasing amounts of lysophosphatides progressively impair membrane configuration thus improving the substrate accessibility for phospholipase A 2 in a second activation step. Finally, the lysophosphatides reach plasma membrane-lytic concentrations by this autocatalytic enzyme activation.

Evidence for aberrant activation of the interleukin-2 autocrine loop by HTLV-1-encoded p40 x and T3/Ti complex triggering

In this study we provide evidence that distinct DNA sequences within the 5′-flanking regions of the genes for interleukin-2 (IL-2) and its receptor (IL-2R) are involved in human T-cell-specific activation of transcription by p40 x, a product of human T cell leukemia virus type I (HTLV-1). The same DNA sequences appear to be responsible for induction of the genes in a T cell line, Jurkat, by mitogens. Although the IL-2 gene sequences are activated by p40 x with much lower efficiency than the IL-2R gene sequences, they are synergistically activated by the p40 x expression and subsequent extracellular stimulation by Concanaval-in-A or anti-T3. We propose a model for two-step activation of the IL-2 autocrine loop in ATL development.

Protein synthesis in rabbit reticulocytes. A study of the roles of Co-eIF-2, Co-eIF-2A80, and GDP in peptide chain initiation.

The roles of Co-eIF-2, Co-eIF-2A80, and GDP in ternary complex and Met-tRNAf X 40 S initiation complex formation were studied. 1) Partially purified eukaryotic initiation factor 2 (eIF-2) (50% pure) preparations contained 0.4-0.6 pmol of bound GDP/pmol of eIF-2. eIF-2 purity was calculated from ternary complex formation in the absence of Mg2+ and in the presence of excess Co-eIF-2. 2) In the absence of Mg2+, approximately 30% of the potentially active eIF-2 molecules formed ternary complexes, and both Co-eIF-2 and Co-eIF-2A80 were equally effective in full activation of the eIF-2 molecules for ternary complex formation. 3) In the presence of Mg2+, approximately 10% of the potentially active eIF-2 molecules formed ternary complexes in the absence of ancillary factors, and the ancillary factors Co-eIF-2A80 and Co-eIF-2 raised the incorporation to 20 and 50% of the eIF-2 molecules, respectively. 4) In the absence of Mg2+, [3H]GDP in preformed eIF-2 X [3H]GDP was readily displaced by GTP during ternary complex formation. 5) In the presence of Mg2+, [3H]GDP remained tightly bound to eIF-2 and ternary complex formation was inhibited. Co-eIF-2, but not Co-eIF-2A80, was effective in promoting [3H]GDP displacement and the former was more effective in promoting ternary complex formation than the latter. 6) eIF-2 X [3H]GDP was converted to eIF-2 X [3H] GTP by incubation in the presence of nucleoside-5'-diphosphate kinase and ATP, but the eIF-2 X [3H]GTP thus formed did not bind Met-tRNAf in the presence of Mg2+ and required exogeneous addition of Co-eIF-2 and GTP for ternary complex formation and GTP displacement. 7) In the absence of Mg2+, the increased ternary complex formed in the presence of eIF-2 X [3H] GDP and Co-eIF-2A80 (with accompanying loss of [3H] GDP) was inactive in a subsequent reaction, which involves Met-tRNAf transfer to 40 S ribosomes (in the presence of Mg2+), and required trace amounts of Co-eIF-2 for such activity. Based on the above observations, we have suggested a two-step activation of eIF-2 molecules by the Co-eIF-2 protein complex for functional ternary complex formation. One of these steps involves the Co-eIF-2A component of Co-eIF-2. This activation results in stimulated Met-tRNAf binding to eIF-2 and is most apparent in the absence of Mg2+ and with aged eIF-2 molecules.(ABSTRACT TRUNCATED AT 400 WORDS)

Fructose-1,6-bisphosphatase from Synechococcus leopoliensis. Substrate-dependent dimer-tetramer interconversion.

Extracts of Synechococcus leopoliensis (Anacystis nidulans) contain two forms of D-fructose-1,6-bisphosphatase (EC 3.1.3.11) previously designated as forms A and B [Gerbling, K.-P., Steup, M., and Latzko, E. (1984) Arch. Microbiol. 137, 109-114]. Form B, which probably represents the major part of the total extractable fructose-1,6-bisphosphatase activity, has been purified to apparent homogeneity. Gel filtration, non-denaturing polyacrylamide gel electrophoresis, and cross-linking with bis(sulfosuccinimidyl)suberate revealed that the fructose-1,6-bisphosphatase B exists in either a dimeric or in a tetrameric subform, depending upon the absence or presence of fructose-1,6-bisphosphate and Mg2+. The dimer--tetramer interconversion was readily reversible. The results provide evidence for a two-step activation of fructose-1,6-bisphosphatase B involving the reduction of the dimeric subform and the subsequent substrate-dependent conversion of the reduced dimer to a reduced tetramer, which is the only catalytically active state. In contrast to form B, no substrate-dependent interconversion was detected with form A from S. leopoliensis.

Photoelectron spectroscopic determination of the structure of (Cs,O) activated GaAs (110) surfaces

p-GaAs (110) surfaces activated to negative electron affinity (NEA) have been examined with photoelectron spectroscopy. A typical activated GaAs surface is found to consist of both a layer of oxygen bonded to GaAs and a (Cs+,O−2) layer. The GaAs-O layer was not anticipated prior to this work. A GaAs-O-Cs dipole plus the polarization of a Cs+-O−2-Cs+ sandwich layer is proposed to explain the NEA condition based on the structure of activated surfaces found in this work. The identification of the O-GaAs bonding layer explains the better yield achieved by the two-step activation process compared to that achieved by a single-step process. Possible optimization of the activation process by forming the O-GaAs layer and the (Cs+,O−2) layer is also discussed. Adsorption of OH from the residual gas in an ultrahigh vacuum chamber is identified as one degradation mechanism of the GaAs photocathodes.

Two-step laser activation of hematoporphyrin derivative

A new photoactivation mechanism of hematoporphyrin derivative is presented. This process, based on the sequential absorption of two photons, leads to the production of cytotoxic radicals of HpD. The cytocidal efficiency is demonstrated by in vitro experiments.

Asymptotic analysis of a premixed laminar flame governed by a two-step reaction

Once properly optimized, single-step chemistry models are able to recover essential characteristics of flames such as burned gases temperature, flame propagation velocity and thickness. Ignition delay values can be reproduced as well. However, an improved description of either the premixed flame inner structure or the ignition process requires the consideration of the multi-step nature of chemical kinetics. Indeed, single-step chemistry models can neither describe properly the internal structure of laminar premixed flames nor the development of ignition processes. These limitations arise because single-step chemistry does not take into account intermediate species, the importance of which is essential. Thus, the present work is aimed at extending the recently-proposed optimized single-step (OSS) framework to multi-step chemistry. To this purpose an unbranched chain reaction with only two consecutive steps relevant to chain initiation and chain termination is considered. The corresponding kinetics model does involve two fictive species, the characteristics of which are optimized together with the two-step chemical scheme parameters, e.g., pre-exponential factors and activation energies. One of this fictive species is relevant to combustion products while the other represents the whole pool of radicals. The chemistry optimization procedure makes use of a well-defined in-house genetic algorithm and the resulting optimized two-step (OTS) model is assessed through comparisons made with data obtained from detailed chemistry computations used as reference. For similar (and even lower) CPU costs, the computations performed with the OTS model put into evidence significant improvements compared to the results obtained with the OSS description.

The Conversion of Prothrombin to Thrombin: V. THE ACTIVATION OF PROTHROMBIN BY FACTOR Xa IN THE PRESENCE OF PHOSPHOLIPID

Abstract As shown previously for activated Factor X and Ca2+ alone, (Esmon, C. T., and Jackson, C. M. (1974) J. Biol. Chem. 249, 7782–7790) prothrombin activation by activated Factor X and Ca2+ in the presence of phospholipid also proceeds via Intermediate 2 and Fragment 1·2, i.e. [see PDF for equation] Unless rapidly inactivated, thrombin cleaves Fragment 1·2 into two smaller activation products, i.e. Fragment 1·2 (Thrombin)/→ Fragment 1 + Fragment 2 2) The direct and concomitant formation of Intermediate 2 and Fragment 1·2 was demonstrated by activating prothrombin in the presence of iPr2P-F. In the absence of this inhibitor, thrombin, in addition to catalyzing the reaction of Equation 2, cleaves Fragment 1 from prothrombin and in so doing eliminates the possibility of observing phospholipid catalysis from all but the earliest stages of reaction (Gitel, S. N., Owen, W. G., Esmon, C. T., and Jackson, C. M. (1973) Proc. Nat. Acad. Sci. U. S. A. 70, 1344–1348). The reaction of Equation 1 was shown to occur independent of any special effect of diisopropyl fluorophosphate (iPr2P-F) by a direct demonstration of Intermediate 2 and Fragment 1·2 formation from tritium-labeled prothrombin. Both the formation of Intermediate 2 and its conversion to thrombin are accelerated by phospholipid. However, in the presence of phospholipid, Intermediate 2 was found to satisfy the necessary condition for an intermediate on a sequential activation path, i.e. that its rate of conversion to thrombin be greater than or equal to the rate of prothrombin conversion to thrombin, if and only if, Fragment 1·2 were also present in the activation mixture. In view of this observation, the true activation intermediate of the phospholipid-catalyzed pathway must consist of both Intermediate 2 and Fragment 1·2. Fragment 1 plus Fragment 2 was not able to substitute for Fragment 1·2. Intermediate 2 and Fragment 1·2 were demonstrated to associate noncovalently to form a product with gel electrophoretic properties indistinguishable from those of prothrombin. This association of Fragment 1·2 with Intermediate 2 apparently provides the mechanism by which Intermediate 2 can be retained on the phospholipid surface and thus be readily accessible to the activated Factor X for the final proteolysis which is required to form thrombin. An alternative prothrombin activation pathway which maintains the integrity of the Intermediate 2 and Fragment 1·2 portions of the prothrombin molecule during the two-step activation process was proposed previously (Esmon, C. T., Owen, W. G., and Jackson, C. M. (1974) J. Biol. Chem. 249, 606–611). No unambiguous evidence for the existence of the alternative activation route could be demonstrated and thus the reactions of Equation 1 appear to describe the principal, if not exclusive, pathway of prothrombin activation by Factor Xa, phospholipid, and Ca2+.