Intermediary Reactions Research Articles

Directing Nanoparticle Organization in Response to Diverse Chemical Inputs.

Signaling cascades are crucial for transducing stimuli in biological systems, enabling multiple stimuli to regulate a downstream target with precisely controlled timing and amplifying signals through a series of intermediary reactions. Developing a robust signaling system with such capabilities would be pivotal for programming complex behaviors in synthetic DNA-based molecular devices. However, although "software" such as nucleic acid circuits could potentially be harnessed to relay signals to DNA-based nanostructure hardware, such explorations have been limited. Here, we develop a platform for transducing a variety of stimuli via messenger-mediated reactions to regulate the release and reloading of gold nanoparticles (AuNPs) in a 3D DNA framework. In the first step, an in vitro transcription circuit is engineered to sense and amplify chemical stimuli, including arbitrary DNA sequences and proteins, producing RNA. In the second step, the RNA releases the DNA-coated AuNPs from the DNA framework via a strand displacement reaction. AuNP reloading is controlled by a separate step driven by degradation of the RNA. Our platform holds promise for applications requiring dynamic multiagent control over DNA-based devices, offering a versatile tool for advanced molecular device engineering.

Thermal decomposition of calcium oxalate: beyond appearances

The goal of this study is twofold: to take a fresh look at the decomposition of calcium oxalate and to warn users of thermogravimetric analysis against the hasty interpretation of results obtained. Since the pioneer work of Duval 70 years ago, the scientific community has agreed unanimously as to the decomposition of anhydrous calcium oxalate (CaC2O4) into calcium carbonate (CaCO3) and CO gas, and that of the calcium carbonate into calcium oxide (CaO), and CO2 gas. We will demonstrate how these reactions, simple in appearance, in fact result from a succession of reactive phenomena involving numerous constituents both solid (CaCO3, free carbon) and gaseous (CO2 and CO) produced by intermediary reactions. The mass losses evaluated in the two distinct domains correspond closely to the molar masses of CO and CO2, respectively. The simple mathematical calculation of that mass loss has simply concealed the existence of other reactions, and, most particularly the Boudouard reaction and that of solid phases between CaCO3 and C. It just goes to show that appearances can be deceiving.

Genome-scale modeling and transcriptome analysis of Leuconostoc mesenteroides unravel the redox governed metabolic states in obligate heterofermentative lactic acid bacteria

Obligate heterofermentative lactic acid bacteria (LAB) are well-known for their beneficial health effects in humans. To delineate the incompletely characterized metabolism that currently limits their exploitation, at systems-level, we developed a genome-scale metabolic model of the representative obligate heterofermenting LAB, Leuconostoc mesenteroides (iLME620). Constraint-based flux analysis was then used to simulate several qualitative and quantitative phenotypes of L. mesenteroides, thereby evaluating the model validity. With established predictive capabilities, we subsequently employed iLME620 to elucidate unique metabolic characteristics of L. mesenteroides, such as the limited ability to utilize amino acids as energy source, and to substantiate the role of malolactic fermentation (MLF) in the reduction of pH-homeostatic burden on F0F1-ATPase. We also reported new hypothesis on the MLF mechanism that could be explained via a substrate channelling-like phenomenon mainly influenced by intracellular redox state rather than the intermediary reactions. Model simulations further revealed possible proton-symporter dependent activity of the energy efficient glucose-phosphotransferase system in obligate heterofermentative LAB. Moreover, integrated transcriptomic analysis allowed us to hypothesize transcriptional regulatory bias affecting the intracellular redox state. The insights gained here about the low ATP-yielding metabolism of L. mesenteroides, dominantly controlled by the cellular redox state, could potentially aid strain design for probiotic and cell factory applications.

Investigating the link of ACAD10 deficiency to type 2 diabetes mellitus.

The Native American Pima population has the highest incidence of insulin resistance (IR) and type 2 diabetes mellitus (T2DM) of any reported population, but the pathophysiologic mechanism is unknown. Genetic studies in Pima Indians have linked acyl-CoA dehydrogenase 10 (ACAD10) gene polymorphisms, among others, to this predisposition. The gene codes for a protein with a C-terminus region that is structurally similar to members of a family of flavoenzymes-the acyl-CoA dehydrogenases (ACADs)-that catalyze α,β-dehydrogenation reactions, including the first step in mitochondrial FAO (FAO), and intermediary reactions in amino acids catabolism. Dysregulation of FAO and an increase in plasma acylcarnitines are recognized as important in the pathophysiology of IR and T2DM. To investigate the deficiency of ACAD10 as a monogenic risk factor for T2DM in human, an Acad-deficient mouse was generated and characterized. The deficient mice exhibit an abnormal glucose tolerance test and elevated insulin levels. Blood acylcarnitine analysis shows an increase in long-chain species in the older mice. Nonspecific variable pattern of elevated short-terminal branch-chain acylcarnitines in a variety of tissues was also observed. Acad10 mice accumulate excess abdominal adipose tissue, develop an early inflammatory liver process, exhibit fasting rhabdomyolysis, and have abnormal skeletal muscle mitochondria. Our results identify Acad10 as a genetic determinant of T2DM in mice and provide a model to further investigate genetic determinants for insulin resistance in humans.

Chemical Investigation on Various Aromatic Compounds Polymerization in Low Pressure Helium Plasma

Remarkable properties of plasma polymer films are greatly dependent not only on the chemical structure of precursor but also on the reactor design and the deposition conditions. In many industrial applications it is a challenge to control the plasma polymer structure. In this paper we investigate the chemical transformation of various aromatic compounds, such as activation and fragmentation of substituent-part, aromatic ring opening, during plasma polymerization process. Polymerized films are deposited in a low- frequency capacitively coupled plasma-enhanced chemical vapour deposition reactor, working at low pressure. The chemical composition of plasma-polymerized films is elu- cidated by Fourier-transform infrared spectroscopy, solid-state carbon-13 nuclear magnetic resonance spectroscopy, and X-ray photoelectron spectroscopy. Based on spectroscopic measurements, the intermediary reactions during film growth may be presumed.

Development of an analytical–numerical solution for a steady and axisymmetric turbulent jet diffusion flame for the hydrogen based on a reduced kinetic mechanism

The hydrogen fuel is considered to be an ideal source of energy, because its complete combustion generates no pollutants, only water vapor. Therefore, the hydrogen has been suggested as a clean fuel. A detailed kinetic mechanism for the combustion of hydrogen that comprises eight species (H2,O2,O,OH,H2O,H,HO2 and H2O2) and 20 elementary reactions, was reduced to two-step mechanism for nonpremixed flames involving four reactive species (H2,O2,H,H2O). We performed, for this mechanism, a numerical analysis of the equations, including the velocity, mixture fraction, mass fractions and temperature. To quantify the components of intermediary reactions, the mixture fraction is decomposed into three parts, each part directly related to the mass fraction of each species. The results compare favorably with data in the literature for a jet diffusion flame of 50/50% in volume of H2-N2. The main advantage of the strategy employed here is to decrease the work needed to solve the system of equations of the reactive flow.

Living with high putrescine: expression of ornithine and arginine biosynthetic pathway genes in high and low putrescine producing poplar cells

Arginine (Arg) and ornithine (Orn), both derived from glutamate (Glu), are the primary substrates for polyamine (PA) biosynthesis, and also play important roles as substrates and intermediates of overall N metabolism in plants. Their cellular homeostasis is subject to multiple levels of regulation. Using reverse transcription quantitative PCR (RT-qPCR), we studied changes in the expression of all genes of the Orn/Arg biosynthetic pathway in response to up-regulation [via transgenic expression of mouse Orn decarboxylase (mODC)] of PA biosynthesis in poplar (Populus nigra×maximowiczii) cells grown in culture. Cloning and sequencing of poplar genes involved in the Orn/Arg biosynthetic pathway showed that they have high homology with similar genes in other plants. The expression of the genes of Orn, Arg and PA biosynthetic pathway fell into two hierarchical clusters; expression of one did not change in response to high putrescine, while members of the other cluster showed a shift in expression pattern during the 7-day culture cycle. Gene expression of branch point enzymes (N-acetyl-Glu synthase, Orn aminotransferase, Arg decarboxylase, and spermidine synthase) in the sub-pathways, constituted a separate cluster from those involved in intermediary reactions of the pathway (N-acetyl-Glu kinase, N-acetyl-Glu-5-P reductase, N-acetyl-Orn aminotransferase, N (2)-acetylOrn:N-acetyl-Glu acetyltransferase, N (2)-acetyl-Orn deacetylase, Orn transcarbamylase, argininosuccinate synthase, carbamoylphosphate synthetase, argininosuccinate lyase, S-adenosylmethionine decarboxylase, spermine synthase). We postulate that expression of all genes of the Glu-Orn-Arg pathway is constitutively coordinated and is not influenced by the increase in flux rate through this pathway in response to increased utilization of Orn by mODC; thus the pathway involves mostly biochemical regulation rather than changes in gene expression. We further suggest that Orn itself plays a major role in the regulation of this pathway.

A Multivariate Time Series Approach to Study the Interdependence among O3, NO x , and VOCs in Ambient Urban Atmosphere

A multivariate time series approach vector autoregression (VAR) along with impulse response function and variance decomposition technique has been employed to look into the interrelationship among O3, NO, NO2, and volatile organic compounds (VOCs, namely, benzene, ethylbenzene, toluene, and xylene in the present study) using 3 months long continuous time series data of 1 h average concentration of these pollutants at one of the traffic sites in Delhi, India. It is found that the VAR of order 2 (i.e., past two lagged values of 1 h interval) is sufficient to represent the observed time series at the station studied. The impulse response function and variance decomposition analysis indicate that O3 concentration shows an immediate rise and persists for a longer duration (typically 8–10 h) once the impulse of NO2, benzene, ethylbenzene, or xylene is given in the ambient environment. However, in case of toluene, the reverse effect has been observed. Since O3 forms in the troposphere due to photolysis of NO2, it is not surprising that its impulse triggers O3 formation in the ambient environment. However, in case of VOCs, this has been attributed to their tendency to show higher inclination toward intermediary reactions leading to the formation of O3 rather than their (VOCs) reaction with O3. Among VOCs, only toluene has been observed to show higher inclination toward its reaction with O3. Apart from this, variance decomposition technique also reveals that the relation of NO with NO2 is more important than the relation of NO with O3 creating a conducive atmosphere for O3 formation in the present scenario. Thus, the multivariate time series approach provides significant insight about the role played by the dominant individual VOCs and NOx in influencing the O3 concentration in ambient urban atmosphere whereas a photochemical modeling approach gives an overall view of NOx and VOCs behavior with respect to O3 by using the O3 isopleth technique.

Transformation of Chlorinated Phenolic Compounds by White Rot Fungi

Chlorinated phenolic compounds are among the most abundant recalcitrant wastes produced by the paper and pulp industry, being accumulated in the effluents after secondary treatments. Due to their high toxicity to a wide range of organisms, chlorinated phenolic compounds pose a big concern to human and environmental health. These compounds are produced upon the partial degradation of lignin during bleaching process, and as such they are degradable by white rot fungi, the only organisms capable of degrading lignin to CO2 and H2O. White rot fungi are a group of organisms very suitable for the removal of chlorinated phenolic compounds from the environment. Indeed, they are robust, ubiquitous organisms and may survive also in the presence of high concentrations of various pollutants, even with a low bioavailability. The activity of white rot fungi is mainly due to the action of oxidoreductive enzymes, i.e., laccase, manganese peroxidase and lignin peroxidase, which are deliberately released by fungal cells into their nearby environment. In cases of secondary metabolism and oxidative stress, the fungi produce other intra- and extracellular enzymes to provide substrates for the key enzymes just mentioned. Phanerochaete chrysosporium is one of the most widely studied fungi, for which intermediary products and reactions involved in the degradation of chlorophenols have been identified. Extracellular laccases and peroxidases carry out the first productive step in the oxidation of chlorophenols, forming para-quinones and consequently releasing a chlorine atom. Further degradative steps involving several enzymes and highly reactive, nonspecific redox mediators produced by the fungus render it capable of efficiently degrading several toxic compounds. In soil environment, chlorophenols may be involved in physical–chemical processes such as polymerization and/or adsorption of/on humic substances that may hinder their degradation by microbial cells, even though both processes can result in dehalogenation of the chlorinated compounds. This review analyzes the presence of chlorophenols in the environment, their main chemical and physical properties, and the main processes in their degradation by white rot fungi with particular attention to soil environment. The reactions involved in the process, the intermediary products, the factors that may affect the fungal transformation of chlorophenols, and the possible applications for environmental purposes of both the whole fungal cells and/or their enzymes as isolated catalytic agents are also addressed.

Correlation between uncoupled ATP hydrolysis and heat production by the sarcoplasmic reticulum Ca2+-ATPase: coupling effect of fluoride.

The sarcoplasmic reticulum Ca(2+)-ATPase transports Ca(2+) using the chemical energy derived from ATP hydrolysis. Part of the chemical energy is used to translocate Ca(2+) through the membrane (work) and part is dissipated as heat. The amount of heat produced during catalysis increases after formation of the Ca(2+) gradient across the vesicle membrane. In the absence of gradient (leaky vesicles) the amount of heat produced/mol of ATP cleaved is half of that measured in the presence of the gradient. After formation of the gradient, part of the ATPase activity is not coupled to Ca(2+) transport. We now show that NaF can impair the uncoupled ATPase activity with discrete effect on the ATPase activity coupled to Ca(2+) transport. For the control vesicles not treated with NaF, after formation of the gradient only 20% of the ATP cleaved is coupled to Ca(2+) transport, and the caloric yield of the total ATPase activity (coupled plus uncoupled) is 22.8 kcal released/mol of ATP cleaved. In contrast, the vesicles treated with NaF consume only the ATP needed to maintain the gradient, and the caloric yield of ATP hydrolysis is 3.1 kcal/mol of ATP. The slow ATPase activity measured in vesicles treated with NaF has the same Ca(2+) dependence as the control vesicles. This demonstrates unambiguously that the uncoupled activity is an actual pathway of the Ca(2+)-ATPase rather than a contaminating phosphatase. We conclude that when ATP hydrolysis occurs without coupled biological work most of the chemical energy is dissipated as heat. Thus, uncoupled ATPase activity appears to be the mechanistic feature underlying the ability of the Ca(2+)-ATPase to modulated heat production.

Preparation and characterization of a cell-free system from Chinese hamster ovary cells that translates natural messenger ribonucleic acid and analysis of intermediary reactions

A cell-free system from cultured Chinese hamster ovary cells has been developed, which translates endogenous mRNAs, exogenous natural mRNAs, and synthetic polynucleotide templates. The analysis of most of the reactions involved in initiation, elongation, and termination of protein synthesis can be carried out in this system. The postmitochondrial fraction, containing ribosomal 40 and 60 S subunits, 80 S ribosomes, polysomes, and cytosol proteins, incorporates amino acids into protein. The preparation is capable of recycling endogenous mRNA by initiating protein synthesis on polysomal mRNA, and of initiating protein synthesis on exogenous templates. When endogenous mRNA is degraded with micrococcal nuclease, polysomes are no longer evident and protein synthesis is markedly depended on added mRNA, ATP, GTP, and a nucleoside triphosphate-generating system. Amino acid incorporation is linear for over 2 h, polysomes containing nascent polypeptide chains are reformed and, with time, most of the protein synthesized is released into the media. Gel electrophoretic analysis of the product formed in response to globin mRNA indicates that most of the radioactivity migrates as a single peak, in the region corresponding to globin. Comparison of the electrophoretic pattern obtained from labeled Chinese hamster ovary cells with that from incubations of cell extract and Chinese hamster ovary mRNA indicates that essentially all of the polypeptides formed by the intact cell are synthesized by the cell-free system. Sucrose gradient centrifugation of incubations containing mRNA-depleted extract and [ 35S]methionine, in the absence of added mRNA, is used to detect initiation intermediates in the formation of the [40 S Met-tRNA f] complex and, with added natural mRNA plus cycloheximide, to detect intermediates in the formation of the 80 S initiation complex. Chain elongation reactions are measured by the incorporation of [ 3H]phenylalanine into polyphenylalanine in extracts supplemented with poly(U), or by the formation of nascent polypeptide chains on polysomes with natural mRNA. Chain termination is measured by analyzing the amount of radioactive protein released into the cytosol.

The effect of elevated temperature on protein synthesis in cell-free extracts of cultured Chinese hamster ovary cells

The effect of elevated temperature on the activity of various components involved in protein synthesis was investigated in extracts from cultured Chinese hamster ovary cells. The translation of exogenous mRNA was markedly inhibited by preincubation of the extract for 15 to 20 minutes at 42°C. However, the following intermediary reactions were not affected, or only slightly inhibited, at 42°C: 1) the incorporation of Met-tRNA f into eIF-2·Met-tRNA f·GTP ternary complex; 2) the interaction of the ternary complex with 40S ribosomal subunits to form the 40S preinitiation intermediate; 3) the binding of mRNA and 60S subunits to form the 80S initiation complex; and 4) the reactions catalyzed by elongation factors EF-1 and EF-2. The activity of Met-tRNA synthetase was markedly inhibited, affecting the formation of initiator Met-tRNA f required for the initiation of protein synthesis and the translation of natural mRNA. Other aminoacyl-tRNA synthetases were not significantly affected by the elevated temperature.

Regulation of translation. Analysis of intermediary reactions in protein synthesis in exponentially growing and stationary phase Chinese hamster ovary cells in culture.

Regulation of translation. Analysis of intermediary reactions in protein synthesis in exponentially growing and stationary phase Chinese hamster ovary cells in culture.

Macrophage activation for tumor cytotoxicity: Genetic variation in macrophage tumoricidal capacity among mouse strains

Development of activated tumoricidal macrophages following Mycobacterium bovis, strain BCG infection in vivo or lymphokine treatment in vitro was examined with more than 20 mouse strains. Peritoneal macrophages from 8 of 22 strains failed to develop tumoricidal capacity by 7 days after intraperitoneal BCG infection. Macrophages from 6 of 6 in vivo nonresponder strains also failed to develop tumoricidal capacity after in vitro treatment with lymphokines. Identification of nonresponder mouse strains should provide a useful resource for analysis of intermediary reactions in macrophage activation.

The analysis of intermediary reactions involved in protein synthesis, in a cell-free extract of Saccharomyces cerevisiae that translates natural messenger ribonucleic acid.

The analysis of intermediary reactions involved in protein synthesis, in a cell-free extract of Saccharomyces cerevisiae that translates natural messenger ribonucleic acid.

Macrophage Activation for Tumor Cytotoxicity: Development of Macrophage Cytotoxic Activity Requires Completion of A Sequence of Short-Lived Intermediary Reactions

Abstract Macrophages from endotoxin (LPS)-unresponsive C3H/HeJ mice fail to develop tumoricidal activity after any of several in vivo or in vitro treatments that induce cytotoxicity with cells from LPS-responsive C3H/HeN mice. Under certain conditions, however, these macrophages could become tumoricidal: macrophages from in vivo immune reactions such as those induced by BCG infection, but not cells from irritant-induced peritoneal exudates, developed full cytotoxic capability after further exposure in vitro to microgram per milliliter concentrations of LPS or to supernatants from antigen-stimulated leukocyte cultures (lymphokines). Capacity of macrophages from BCG-infected C3H/HeJ mice to become tumoricidal after LPS exposure was gradually lost with time in culture and was absent by 24 hr. Requirements for at least two activation stimuli for cytotoxic activity with C3H/HeJ macrophages could also be fulfilled entirely in vitro: cells cultured in lymphokines plus LPS were fully tumoricidal; macrophages cultured in either agent alone were not cytotoxic. Similar interactions between lymphokines and LPS could be demonstrated with macrophages from LPS-responsive C3H/HeN mice. Tumoricidal activity by C3H/HeN macrophages cultured in lymphokines plus nanogram per milliliter concentrations of LPS was considerably greater than that by cells cultured in either agent alone. LPS and lymphokines were synergistic in their action on macrophage cytotoxicity. The synergistic effect of LPS on cytotoxicity by lymphokine-activated macrophages was evident after a 15-min pulse and was not dependent upon fluid-phase LPS. Synergistic action, however, was dependent upon 1.) treatment sequence: LPS was active only if given simultaneously with or after lymphokine treatment; LPS exposure before lymphokine treatment was ineffective, and 2.) treatment interval: capacity of lymphokine-treated macrophages to express cytotoxic activity after LPS exposure decayed with time in culture and was lost by 24 hr. Thus, macrophage activation for tumor cytotoxicity in both C3H/HeJ and C3H/HeN mice was the final result of a cascade of short-lived intermediary reactions in a defined sequence. Tumoricidal activity of fully activated cells and responsiveness of each noncytotoxic precursor cell to activation stimuli were all short lived macrophage reactions. Present evidence suggests that none of these reactions was reversible. Completion of each state of macrophage activation was completely dependent upon the simultaneous presence of localized activation signals and macrophage precursors able to respond.

Thermodynamic studies of binary and ternary complexes of pig heart lactate dehydrogenase

The thermodynamics of the reaction catalyzed by pig heart muscle lactate dehydrogenase (LDH; EC1.1.1,27) have been studied in 0,2 M potassium phosphate buffer, pH 7, over the temperature range of 10 to 35 degrees C by using oxamate and oxalate to simulate the corresponding reactions of the substrates pyruvate and lactate, respectively. The various complexes formed are characterized by Gibbs free energies, enthalpies, and entropies. The Gibbs free energies were determined by equilibrium dialysis investigations, fluorescence titrations, and ultraviolet difference spectroscopy, while the reaction enthalpies stem from direct calorimetric measurements, Formulas are given for both the temperature dependence of the equilibrium constants and the variation with temperature of the enthalpies involved in the four reactions between LDH and NADH or NAD, LDH-NADH and oxamate, and LDH-NAD and oxalate. All reactions show a marked negative temperature coefficient, deltacp, of the binding enthalpies indicating partial refolding to be associated with binary and ternary complex formation. This interpretation appears very probable in view of recent x-ray crystallographic studies on lactate dehydrogenase from dogfish, which demonstrate a volume decrease to occur on binding of oxamate to the LDH-NADH complex. The validity of the thermodynamic parameters, as derived with substrate analogues, for the actual catalytic reaction, gains strong support from the agreement between the sum of the heats involved in the four intermediary reactions reported in this study and direct determinations of the overall enthalpy associated with the catalytic process published in the literature.

Equilibrium relations between the oxidation-reduction reactions and the adenosine triphosphate synthesis in suspensions of isolated liver cells.

1. The redox state of cytochrome c, cytochrome a and the mitochondrial NAD couple, and the phosphorylation state of the adenine nucleotides, were measured in suspensions of isolated rat liver cells. 2. The DeltaG for the transfer of two electrons from the mitochondrial NAD to the cytochrome c couple is calculated to be 104kJ (24.8kcal). 3. The DeltaG associated with the synthesis of ATP at the measured phosphorylation state is calculated to be 95kJ (22.7kcal)/2mol of ATP. 4. The near equality of DeltaG of the electron-transport process and DeltaG required for ATP synthesis indicates near-equilibrium between the mitochondrial respiratory chain and the extramitochondrial phosphorylation state. 5. The existence of near-equilibrium in the coupled reactions implies that the respiratory activity depends on the ratio [ATP]/[ADP][P(i)] and not on the concentrations of the individual reactants. 6. If the overall system of oxidative phosphorylation is at near-equilibrium, all intermediary reactions must also be at equilibrium. Hence if the intramitochondrial and extramitochondrial phosphorylation states are indeed different, it follows that any differences in the activities of ATP, ADP and P(i) must be coupled to ion gradients and/or potentials across the inner mitochondrial membrane in such a way that translocation occurs without loss of free energy. 7. The metabolic state of the mitochondria in the cell can be defined by the turnover number of the cytochromes, the cytoplasmic phosphorylation state, and the oxidation-reduction potential of the NAD couple, rather than by the availability of ADP, substrate and O(2).

Intermediary reactions in the water-splitting part of photosynthesis : Bultemann, V., H. Ruppel and H. T. Witt, Technischen Universitat, Berlin, reprint, Nature, November 1964, 7 p. illus.

Intermediary reactions in the water-splitting part of photosynthesis : Bultemann, V., H. Ruppel and H. T. Witt, Technischen Universitat, Berlin, reprint, Nature, November 1964, 7 p. illus.

Androgenic control of nucleic acid and protein synthesis in male accessory genital organs.

A survey is given of experimental studies on the influence of treatment with androgenic hormones in vivo on various intermediary reactions involved in ribonucleic acid (RNA) and protein synthesis in the prostate gland and seminal vesicle, with particular reference to the control of the growth and functional differentiation of these organs by testosterone and related steroids. Studies on the influence of androgens on RNA metabolism and protein biosynthesis in mouse kidney, certain muscles, and some other extragenital tissues are also considered.