Complex Biochemical Reactions Research Articles

The C-terminal Domain of the DNA Polymerase Catalytic Subunit Regulates the Primase and Polymerase Activities of the Human DNA Polymerase α-Primase Complex

The initiation of DNA synthesis during replication of the human genome is accomplished primarily by the DNA polymerase α-primase complex, which makes the RNA-DNA primers accessible to processive DNA pols. The structural information needed to understand the mechanism of regulation of this complex biochemical reaction is incomplete. The presence of two enzymes in one complex poses the question of how these two enzymes cooperate during priming of DNA synthesis. Yeast two-hybrid and direct pulldown assays revealed that the N-terminal domain of the large subunit of primase (p58N) directly interacts with the C-terminal domain of the catalytic subunit of polα (p180C). We found that a complex of the C-terminal domain of the catalytic subunit of polα with the second subunit (p180C-p70) stimulated primase activity, whereas the whole catalytically active heterodimer of polα (p180ΔN-p70) inhibited RNA synthesis by primase. Conversely, the polα catalytic domain without the C-terminal part (p180ΔN-core) possessed a much higher propensity to extend the RNA primer than the two-subunit polα (p180ΔN-p70), suggesting that p180C and/or p70 are involved in the negative regulation of DNA pol activity. We conclude that the interaction between p180C, p70, and p58 regulates the proper primase and polymerase function. The composition of the template DNA is another important factor determining the activity of the complex. We have found that polα activity strongly depends on the sequence of the template and that homopyrimidine runs create a strong barrier for DNA synthesis by polα.

Universal dynamical properties preclude standard clustering in a large class of biochemical data.

Clustering of chemical and biochemical data based on observed features is a central cognitive step in the analysis of chemical substances, in particular in combinatorial chemistry, or of complex biochemical reaction networks. Often, for reasons unknown to the researcher, this step produces disappointing results. Once the sources of the problem are known, improved clustering methods might revitalize the statistical approach of compound and reaction search and analysis. Here, we present a generic mechanism that may be at the origin of many clustering difficulties. The variety of dynamical behaviors that can be exhibited by complex biochemical reactions on variation of the system parameters are fundamental system fingerprints. In parameter space, shrimp-like or swallow-tail structures separate parameter sets that lead to stable periodic dynamical behavior from those leading to irregular behavior. We work out the genericity of this phenomenon and demonstrate novel examples for their occurrence in realistic models of biophysics. Although we elucidate the phenomenon by considering the emergence of periodicity in dependence on system parameters in a low-dimensional parameter space, the conclusions from our simple setting are shown to continue to be valid for features in a higher-dimensional feature space, as long as the feature-generating mechanism is not too extreme and the dimension of this space is not too high compared with the amount of available data. For online versions of super-paramagnetic clustering see http://stoop.ini.uzh.ch/research/clustering. Supplementary data are available at Bioinformatics online.

Single Case Studies as Seeds

Single case studies are research studies of single participants. They explore new ideas and can suggest extensions in methods and for treatment (Yin, 1984). In this article the case study refers only to conditions observed and is limited to what was observed from these comparisons between normal function and patterns of specific conditions. This does not reflect the theory of a formal case study but rather it is an attempt to show the case study and simple computer modelling as learning tools in a complex environment. Critics of case study methodology cite small case numbers as not having grounds for establishing reliability or generalization of findings and that the intensity of exposure needed for thorough case studies could bias the research findings subtracting from researcher objectivity. Early computer models were used to simulate the function of the brain and provide partial answers. They provided insight into the understanding of complex function. In recent history, computer models and case studies have been cast aside in favor of live brain imaging and complex biochemical reactions. It is good to remember that these tools brought us to the place of knowledge we enjoy today and have enlarged diagnostic and treatment choices. They are still valuable and inexpensive methods that can impact the imaginations of neuroscientists and kindle their passions to solve the complexities of the human mind one problem at a time. Greater rigor can be easily maintained by adopting a format whereby a patient would be assessed by a fully standardized neuropsychological battery and the performance then compared to large sample of normative data. The formal study is important for generalization of findings across conditions and can be applied once normative data has been collected as a basis for comparison.

Stochastic modelling of biochemical systems of multi-step reactions using a simplified two-variable model

BackgroundA fundamental issue in systems biology is how to design simplified mathematical models for describing the dynamics of complex biochemical reaction systems. Among them, a key question is how to use simplified reactions to describe the chemical events of multi-step reactions that are ubiquitous in biochemistry and biophysics. To address this issue, a widely used approach in literature is to use one-step reaction to represent the multi-step chemical events. In recent years, a number of modelling methods have been designed to improve the accuracy of the one-step reaction method, including the use of reactions with time delay. However, our recent research results suggested that there are still deviations between the dynamics of delayed reactions and that of the multi-step reactions. Therefore, more sophisticated modelling methods are needed to accurately describe the complex biological systems in an efficient way.ResultsThis work designs a two-variable model to simplify chemical events of multi-step reactions. In addition to the total molecule number of a species, we first introduce a new concept regarding the location of molecules in the multi-step reactions, which is the second variable to represent the system dynamics. Then we propose a simulation algorithm to compute the probability for the firing of the last step reaction in the multi-step events. This probability function is evaluated using a deterministic model of ordinary differential equations and a stochastic model in the framework of the stochastic simulation algorithm. The efficiency of the proposed two-variable model is demonstrated by the realization of mRNA degradation process based on the experimentally measured data.ConclusionsNumerical results suggest that the proposed new two-variable model produces predictions that match the multi-step chemical reactions very well. The successful realization of the mRNA degradation dynamics indicates that the proposed method is a promising approach to reduce the complexity of biological systems.

DNA Double-Strand Break Repair at −15°C

ABSTRACT The survival of microorganisms in ancient glacial ice and permafrost has been ascribed to their ability to persist in a dormant, metabolically inert state. An alternative possibility, supported by experimental data, is that microorganisms in frozen matrices are able to sustain a level of metabolic function that is sufficient for cellular repair and maintenance. To examine this experimentally, frozen populations of Psychrobacter arcticus 273-4 were exposed to ionizing radiation (IR) to simulate the damage incurred from natural background IR sources in the permafrost environment from over ∼225 kiloyears (ky). High-molecular-weight DNA was fragmented by exposure to 450 Gy of IR, which introduced an average of 16 double-strand breaks (DSBs) per chromosome. During incubation at −15°C for 505 days, P. arcticus repaired DNA DSBs in the absence of net growth. Based on the time frame for the assembly of genomic fragments by P. arcticus , the rate of DNA DSB repair was estimated at 7 to 10 DSBs year −1 under the conditions tested. Our results provide direct evidence for the repair of DNA lesions, extending the range of complex biochemical reactions known to occur in bacteria at frozen temperatures. Provided that sufficient energy and nutrient sources are available, a functional DNA repair mechanism would allow cells to maintain genome integrity and augment microbial survival in icy terrestrial or extraterrestrial environments.

Oxidative Processes in Muscle Systems and Fresh Meat: Sources, Markers, and Remedies.

Oxidation of meat occurs under postmortem conditions and is inevitable. This oxidation includes the biochemical changes in meat leading to changes in color pigments and lipids. As a consequence, color deteriorates, and undesirable flavors and rancidity develop in meat thereby impacting on consumer appeal and satisfaction. Across carcasses, there is variation in the rate at which muscle undergoes chemical reactions under postmortem conditions that reflect inherent variation at the biochemical level. It is expected that this underlying biochemical variation will be reflected in living muscle through oxidative processes. The oxidative process of muscle tissues will vary according to an animal's immunity status, temperament, and ability to cope with stress, with all these affected by nutrition, genetics, management practices, and environmental conditions (hot and cold seasons). Identification of biomarkers that indicate the oxidative status levels of animals or muscle tissues in vivo could provide insight as to how the muscle will respond to the anoxic conditions that produce undesirable results in meat. This review outlines the potential use of 1 group of biomarkers, the isoprostanes, in the context of complex biochemical reactions relating to oxidative processes that take place in the biological systems of live animals (in vivo) and subsequently in meat (in vitro).

DEVELOPMENT OF MODIFIED ATMOSPHERE PACKAGES ON THE QUALITY OF SICILIAN KALE (BRASSICA OLERACEA VAR. ACEPHALA) SHOOTS

Since some decades, and also in the present global economic crisis, the request for fresh-cut products has showed an increasing trend, and its productive chain represent one of the most remunerative activity of agriculture. In this frame we pointed our attention to diversify fresh-cut vegetable items by several minor and/or underutilized crops and in some case by wild species gathered and utilized as vegetables in Sicily. Kale (Brassica oleracea var. acephala) shoots seem to be of great interest for fresh-cut production both for its environmental friendly growing techniques and nutraceutical properties, as it is rich in antioxidants compounds as ascorbic acid, total polyphenols, carotenoids and glucosinolates. The use of low level of oxygen (O2) and of high level of carbon dioxide (CO2) atmosphere appeared useful for maintain quality and extend shelf-life for several vegetables. In this condition the plant reduces respiration process that involves a complex biochemical reactions, delayed ripening and senescence and also reduces fungal development. Besides, refrigeration is important for elongate the shelf-life but modified atmosphere packaging (MAP) is an important complementary technique to apply. We studied a kale Sicilian type evaluating three modified atmospheres (air, 70% N2:30% CO2; 100% CO2), three temperature levels (0, 4 and 8°C) during three storage times (0, 3 and 7 days). Every day, the percentages of oxygen and carbon dioxide, colour parameters (CIE L*, a* and b*) and dry weight of the shoots were monitored. Results showed the best shoots quality utilizing 70% N2:30% CO2 storage atmosphere at 4°C, the product is kept in good condition for all seven days. This study has improved knowledge about the respiration process and variation of color of kale shoots in relation to modified atmospheres packaging, temperature and time.

Modeling Gene Regulation in Graded Hypoxia Using Continuous-Time Markov Chains

Hypoxia inducible factor (HIF) is the main protein in hypoxia pathway. The response of HIF to changes of oxygen pressure is regulated by 2 oxygen sensors, prolyl hydroxylase (PHD) and factor inhibiting HIF (FIH). Studies have shown that biochemical reactions at molecular level actually exhibit stochastic and random behaviors. Modeling biochemical reactions using purely deterministic method, therefore, ignore these characteristics. Hence, we use stochastic modeling using CTMC to model this regulation. Nevertheless, the use of pure CTMC on complex biochemical reaction networks, such as hypoxia, results in an infeasible computation time and typically requires very large memory. Therefore, we use a numerical hybrid method that combines pure CTMC and deterministic methods. The purpose is to reduce time complexity and to obtain a better accuracy than deterministic method. Using this model, we can observe that an increase of oxygen pressure results in a decrease in the amount of HIF and that oxygen sensor FIH only inhibits C-TAD activity. The model is also able to classify 84% genes that were observed DOI: http://dx.doi.org/10.11591/ij-ict.v2i2.3845

OsbZIP58, a basic leucine zipper transcription factor, regulates starch biosynthesis in rice endosperm

Starch composition and the amount in endosperm, both of which contribute dramatically to seed yield, cooking quality, and taste in cereals, are determined by a series of complex biochemical reactions. However, the mechanism regulating starch biosynthesis in cereal seeds is not well understood. This study showed that OsbZIP58, a bZIP transcription factor, is a key transcriptional regulator controlling starch synthesis in rice endosperm. OsbZIP58 was expressed mainly in endosperm during active starch synthesis. osbzip58 null mutants displayed abnormal seed morphology with altered starch accumulation in the white belly region and decreased amounts of total starch and amylose. Moreover, osbzip58 had a higher proportion of short chains and a lower proportion of intermediate chains of amylopectin. Furthermore, OsbZIP58 was shown to bind directly to the promoters of six starch-synthesizing genes, OsAGPL3, Wx, OsSSIIa, SBE1, OsBEIIb, and ISA2, and to regulate their expression. These findings indicate that OsbZIP58 functions as a key regulator of starch synthesis in rice seeds and provide new insights into seed quality control.

An unknown oxidative metabolism substantially contributes to soil CO<sub>2</sub> emissions

Abstract. The respiratory release of CO2 from soils is a major determinant of the global carbon cycle. It is traditionally considered that this respiration is an intracellular metabolism consisting of complex biochemical reactions carried out by numerous enzymes and co-factors. Here we show that the endoenzymes released from dead organisms are stabilised in soils and have access to suitable substrates and co-factors to permit function. These enzymes reconstitute an extracellular oxidative metabolism (EXOMET) that may substantially contribute to soil respiration (16 to 48% of CO2 released from soils in the present study). EXOMET and respiration from living organisms should be considered separately when studying effects of environmental factors on the C cycle because EXOMET shows specific properties such as resistance to high temperature and toxic compounds.

189 Structural articulation of biochemical reactions using altered bonds and iterative topology switching

Structural and dynamic trajectories of biochemical reactions catalyzed by large macromolecular complexes are difficult and expensive to characterize experimentally. Predicting them thoroughly using quantum mechanics/molecular mechanics (QM/MM) approaches is possible, but requires significant computational resources. Atomic detail structural snapshots of individual states obtained through crystallography may also not always be in a conformation conducive to the reaction, presenting a hurdle for choosing the appropriate starting structures for the QM/MM calculations. The geometric displacements for nuclei that change their bonding during chemical reactions are usually not large, when starting from appropriate reactive conformations. If we hypothesize that there is no need for these rearrangements to be represented in electronic detail to capture the response of the surrounding macromolecular environment, MM energy functions should provide a relatively accurate description of this structural reorganization. In the present study, a strategy called altered bonds and iterative topology swapping with MM models is used to obtain reasonable atomic detail trajectories for complex biochemical reactions. The implementation, advantages, and disadvantages of this strategy are illustrated using a symmetric model proton transfer reaction. This application is expanded to describe a similar proton-transfer reaction in a complex macromolecular environment. The strategy is then utilized to visualize a complex biochemical reaction representing a critical step in the central dogma of biology, namely addition of a deoxyribonucleotide to a DNA strand by a DNA polymerase. This strategy reduces the computational cost of obtaining reasonable biochemical reaction trajectories within complex macromolecular environments by orders of magnitude as compared to QM/MM simulations, thus rapidly identifying good structural starting points for such simulations. Its easy implementation allows the direct application of most computational methods designed for MM models towards understanding reactions involving covalent changes. It also enables a reasonable structural prediction for all reactant, transition, intermediate, and product states for any biochemical reaction for which only one of these states is known. Its high-throughput application offers the promise of delineating, dynamically and in atomic detail, any biochemical reaction catalyzed by a macromolecular catalyst whose structure is either known or can be accurately modeled.

Abnormal fermentations in table-olive processing: microbial origin and sensory evaluation.

The process of transformation of table olives from tree to table is the result of complex biochemical reactions that are determined by the interactions between the indigenous microflora of the olives, together with a variety of contaminating microrganisms from different sources [fiber-glass fermenters, polyvinyl chloride (PVC) tanks, pipelines, pumps, and water], with the compositional characteristics of the fruit. One of the most important aspects of improving the quality of table olives is the use of selected microorganisms to drive the fermentation. These can supplant the indigenous microflora and, in particular, the complementary microflora that are responsible for spoilage of canned olives. In this context, from a technological point of view, a well-characterized collection of microrganisms (lactic acid bacteria, yeast) that can be isolated from the matrix to be processed (the olive fruit) will provide the basis for the development of starter culture systems. These cultures can be fully compatible with the typical products and will guarantee high quality standards. Inoculation of the brine with such selected starter cultures will reduce the probability of spoilage, and help to achieve an improved and more predictable fermentation process. Control of the fermentation processes can thus occur through chemical, chemico-physical and microbiological approaches, and since 2008, also through organoleptic evaluation (COI/OT/MO/Doc. No 1. Method for the sensory analysis of table olives). This last has established the necessary criteria and procedures for sensory analysis of the negative, gustatory and kinaesthetic sensations of table olives, which can also be attributed to abnormal proliferation of microrganisms. It also sets out the system for commercial classification, through assessment of the median of the defect predominantly perceived.

Beer Brewing Control System Based on DS18B20

Beer brewing process has a very high demand for temperature. The beer fermentation process is the core, which is a very complex biochemical exothermic reaction. The temperature of the controlled object has a variability and uncertainty. Fermentation temperature determines the quality of the product. Therefore, we must control the temperature strictly during fermentation. The paper introduces the beer fermentation temperature measurement and control by using the DS18B20 temperature measurement system. Through the system we can significantly improve the technical parameters of the fermentation temperature, which can significantly improve the quality of beer. This system has a wide range of applications.

Global injectivity and multiple equilibria in uni- and bi-molecular reaction networks

Dynamical system models of complex biochemical reaction networks are high-dimensional, nonlinear, and contain many unknown parameters.The capacity for multiple equilibria in such systems plays a key rolein important biochemical processes. Examples show that there is a verydelicate relationship between the structure of a reaction network andits capacity to give rise to several positive equilibria. In thispaper we focus on networks of reactions governed by mass-actionkinetics. As is almost always the case in practice, we assume that noreaction involves the collision of three or more molecules at the sameplace and time, which implies that the associated mass-actiondifferential equations contain only linear and quadratic terms. Wedescribe a general injectivity criterion for quadratic functions ofseveral variables, and relate this criterion to a network's capacityfor multiple equilibria. In order to take advantage of this criterionwe look for explicit general conditions that imply non-vanishing ofpolynomial functions on the positive orthant.In particular, we investigate in detail the case of polynomials withonly one negative monomial, and we fully characterize the case ofaffinely independent exponents.We describe several examples, including an example that shows howthese methods may be used for designing multistable chemical systemsin synthetic biology.

Regularization of inverse problems to determine transcription factor profiles from fluorescent reporter systems

Signal transduction pathways are characterized by complex biochemical reactions which involve a large number of proteins. The availability and quality of experimental data pose challenges for identifying the role of individual proteins in these pathways. To address this issue, this article formulates and solves an inverse problem to determine the dynamics of transcription factors from fluorescence intensity measurements of green fluorescent protein (GFP) reporter systems. In the presented approach, a model describing transcription and translation of GFP is discretized and concentrations of transcription factor are estimated at discrete time points. Unlike previous studies, this approach has no restrictions with regard to a particular shape of the profiles. However, the resulting inverse problem is ill-conditioned and requires the use of regularization techniques. Two regularization methods—truncated singular value decomposition and Tikhonov regularization—are investigated in this work and the characteristics of the results obtained are discussed in detail. © 2012 American Institute of Chemical Engineers AIChE J, 2012

Cell-free protein synthesis from a single copy of DNA in a glass microchamber

To achieve a cell-mimetic reaction environment, we fabricated and tested quartz microchambers for conducting protein synthesis using an in vitro transcription and translation system, the PURE system. By introducing a glass microchamber and blocking the surface of the chamber with amino acids, the concentration of the synthesized marker protein (green fluorescent protein, GFP) was significantly improved compared to that in the poly(dimethylsiloxane) (PDMS) microchamber. The concentration was below the detection limit in the PDMS microchambers, whereas the glass microchambers yielded 700 nM GFP, representing 41% of the bulk reaction. There was no detectable difference when the GFP synthesis was performed in microchambers with sizes ranging from 40 fL to 7 pL, indicating that the present microchamber system can serve as a cell-sized test tube with a variable reaction volume. Finally, we demonstrated that two different proteins, GFP and β-galactosidase, can be expressed from single genes in our experimental setup. Quantized and distinctive signals from proteins synthesized from 0, 1, or 2 copies of genes were obtained. The microchamber presented here can be utilized not only to study the effects of compartment volume on protein synthesis but also for the comprehensive analysis of complex biochemical reactions in cell-mimetic environments.

Using enzymatic numerical P systems for modeling mobile robot controllers

P systems (PSs) are powerful computing models based on the structure of a biological cell and on the way chemicals interact in complex biochemical reactions which take place in various compartments (or membranes) of the cell. A lot of interest has been focused on developing various forms of PSs, from cell-like to tissue-like structures. Most of the research effort has been concentrated on symbolical PSs. Numerical P systems (NPSs) have been introduced in 2006 for possible applications in economics and business processes but no other structures and applications concerning this type of PSs have been provided since then. This paper proposes a new class of NPSs, in which enzyme-like variables allow the existence of more than one production function in each membrane, while keeping the deterministic nature of the system. The way this new type of deterministic NPSs works and a possible use of it for modeling mobile robot controllers are detailed.

Sequential Injection Analysis for Optimization of Molecular Biology Reactions

In order to automate the optimization of complex biochemical and molecular biology reactions, we developed a sequential injection analysis (SIA) device and combined this with a design of experiment (DOE) algorithm. This combination of hardware and software automatically explores the parameter space of the reaction and provides continuous feedback for optimizing reaction conditions. As an example, we optimized the endonuclease digest of a fluorogenic substrate and showed that the optimized reaction conditions also applied to the digest of the substrate outside of the device and to the digest of a plasmid. The sequential technique quickly arrived at optimized reaction conditions with less reagent use than a batch process (such as a fluid handling robot exploring multiple reaction conditions in parallel) would have. The device and method should now be amenable to much more complex molecular biology reactions whose variable spaces are correspondingly larger.

Radical break-up, blissful make-up

The radical SAM superfamily of enzymes provides, yet again, fertile ground for the discovery of amazing new biochemical transformations. Strong evidence is now presented for an unprecedented radical fragmentation-recombination of L-tryptophan to a derivative that is incorporated into the complex antibiotic nosiheptide, highlighting the versatility of radical mechanisms for complex biochemical reactions.

Application of improved dynamic matrix control algorithm to fermentation temperature control

Beer fermentation is a kind of complex biochemical reaction,the temperature control of which has the feature of large time delay,and it is difficult to establish an accurate mathematical model for this plant as the complex mechanism and the changing environment.The conventional control algorithms always cannot act well in this kind of system,especially while there are several unpredictable interferences.In order to solve these problems,a new prediction control strategy for fermentation temperature was proposed based on Dynamic Matrix Control (DMC) which had been improved by the time optimal control.The application results show that this strategy exerts a good effect on the fermentation temperature control and quickly suppresses the interferences.