Thermodynamic Constraints in Kinetic Modeling: Thermodynamic‐Kinetic Modeling in Comparison to Other Approaches

Kinetic models of reaction networks may easily violate the laws of thermodynamics and the principle of detailed balance. In large network models, the constraints that are imposed by these laws are particularly difficult to address. This hinders modeling of biochemical reaction networks. Thermodynamic‐kinetic modeling is a method that provides a thermodynamically sound and formally appealing way for deriving dynamic model equations of reaction systems. State variables of this approach are thermokinetic potentials that describe the ability of compounds to drive a reaction. A compound has a parameter called capacity, which is the ratio of its concentration and thermokinetic potential. A reaction is described by its resistance which is the ratio of the thermokinetic driving force and flux. In these aspects, the formalism is similar to the modeling formalism for electrical networks and an analogous graphical representation is possible. The thermodynamic‐kinetic modeling formalism is equivalent to the traditional kinetic modeling formalism with the exception that it is not possible to build thermodynamically infeasible models. Here, the thermodynamic‐kinetic modeling formalism is reviewed, compared to other approaches, and some of its advantages are worked out. In contrast to other approaches, thermodynamic‐kinetic modeling does not rely on an explicit enumeration of stoichiometric cycles. It is capable of describing rate laws far from equilibrium. Further, the parameterization by capacities and resistances is particularly intuitive and powerful.     

Coupling of Proteolysis to ATP Hydrolysis upon Escherichia coliLon Protease Functioning: II. Hydrolysis of ATP and Activity of the Enzyme Peptide Hydrolase Sites

The absence of direct correlation between the efficiency of functioning of ATPase and peptide hydrolase sites of Lon protease was revealed. It was shown that Lon protease is an allosteric enzyme, in which the catalytic activity of peptide hydrolase sites is provided by the binding of nucleotides, their magnesium complexes, and free magnesium ions in the enzyme ATPase sites. It was revealed that the ADP–Mg complex, an inhibitor of the native enzyme, is an activator of the Lon-K362Q (the Lon protease mutant in the ATPase site). Variants of functional contacts between different sites of the enzyme are considered. It was established that two ways of signal transduction from the ATPase sites to peptide hydrolase ones exist in the Lon protease oligomer--intra- and intersubunit ways. The enzyme ATPase sites are suggested to be located in the areas of the complementary surfaces of subunits. It is hypothesized that upon degradation of protein substrates by the E. coliLon protease in vivoATP hydrolysis acts as a factor of limitation of the enzyme degrading activity.     

Cytoplasmic incompatibilities in the mosquito Culex pipiens: How to explain a cytotype polymorphism?*

Although cytoplasmic incompatibilities have been used as a means of eradicating the mosquito Culex pipiens, the population dynamics of these sterilities in relation to the coexistence of multiple incompatible cytotypes in a single area has not been investigated, except in the case of two unidirectionally incompatible cytotypes. An analytical model of the evolution of n cytotypes in an infinite panmictic population has been developed in order to investigate polymorphic equilibrium. A necessary criterion for the stability of such an equilibrium is established; it is shown that a stable polymorphism cannot exist between incompatible cytotypes. This result is discussed in the light of population dynamics and genetics of Culex pipiens, and of our present knowledge on incompatibilities. The consequences of a geographic structuring and of homogamy are considered. A careful reconsideration of previous experimental results disclosed probable nuclear effects and a serious experimental weakness: with the common procedure of backcrossing hybrid females to males of constant genotype it is not possible to rule out probable nuclear effects with paternal expression. It is concluded that incompatibilities in Culex pipiens may have a nuclear-cytoplasmic determinism.     

Hydrolysis of ATP by aminoglycoside 3'-phosphotransferases: an unexpected cost to bacteria for harboring an antibiotic resistance enzyme

Aminoglycoside 3'-phosphotransferases (APH(3')s) are common bacterial resistance enzymes to aminoglycoside antibiotics. These enzymes transfer the gamma-phosphoryl group of ATP to the 3'-hydroxyl of the antibiotics, whereby the biological activity of the drugs is lost. Pre-steady-state and steady-state kinetics with two of these enzymes from Gram-negative bacteria, APH(3')-Ia and APH(3')-IIa, were performed. It is demonstrated that these enzymes in both ternary and binary complexes facilitate an ATP hydrolase activity (ATPase), which is competitive with the transfer of phosphate to the antibiotics. Because these enzymes are expressed constitutively in resistant bacteria, the turnover of ATP is continuous during the lifetime of the organism both in the absence and the presence of aminoglycosides. Concentrations of the enzyme in vivo were determined, and it was estimated that in a single generation of bacterial growth there exists the potential that this activity would consume as much as severalfold of the total existing ATP. Studies with bacteria harboring the aph(3')-Ia gene revealed that bacteria are able to absorb the cost of this ATP turnover, as ATP is recycled. However, the cost burden of this adventitious activity manifests a selection pressure against maintenance of the plasmids that harbor the aph(3')-Ia gene, such that approximately 50% of the plasmid is lost in 1500 bacterial generations in the absence of antibiotics. The implication is that, in the absence of selection, bacteria harboring an enzyme that catalyzes the consumption of key metabolites could experience the loss of the plasmid that encodes for the given enzyme.     

Proteolysis Coupled to ATP Hydrolysis: Regulation of the Activity of Proteolytic Sites of Lon Protease from Escherichia coli

Regulation of activity of the proteolytic sites of Lon protease was studied. It was found that ATP–Mg has the properties of a noncompetitive activator of peptidase sites. The processive mechanism of the hydrolysis of protein substrates by Lon protease was experimentally confirmed under the conditions of ATP hydrolysis. It was shown that the oligomeric state of the enzyme is the necessary prerequisite for the processive proteolysis by native Lon protease. The study of the properties of the mixed mutant Lon-K362Q/S679A confirmed the existence of intra- and intersubunit pathways of signal transduction from the ATPase to proteolytic sites. The mutual influence of substrates of Lon protease was studied, and the existence of cooperative interactions between the peptidase sites in the oligomeric enzyme was suggested.     

Individual Nuclei Differ in Their Sensitivity to the Cytoplasmic Inducers of DNA Synthesis: Implications for the Origin of Cell Cycle Variability

Nuclei of multinucleate cells generally initiate DNA synthesis simultaneously, suggesting that the timing of DNA synthesis depends upon the appearance of a cytoplasmic signal. In contrast, intact nuclei from quiescent mammalian cells initiate DNA synthesisasynchronouslyin cell-free extracts ofXenopuseggs, despite the common environment. Here we show that the two nuclei of permeabilized binucleate cells enter DNA synthesis coordinately in egg extracts, as they doin vivo,with different pairs of nuclei initiating replication at different times. This indicates that the two nuclei of a binucleate cell are identical in their sensitivity to the inducers of DNA synthesis in egg extracts; this sensitivity varies in general between the nuclei of unrelated cells. The asynchrony of DNA synthesis shown by unrelated nuclei in egg extracts is therefore not an artifact of the cell-free system but a reflection of genuine differences preexisting within the intact cell. Evidence that these differences between nuclei are responsible for a substantial fraction of G₁variability in living cells is presented.     

<Emphasis Type="Italic">Tequilana weber</Emphasis> Agave Bagasse Enzymatic Hydrolysis for the Production of Fermentable Sugars: Oxidative-Alkaline Pretreatment and Kinetic Modeling

As a way to mitigate the harmful effects of fossil fuel utilization, the use of second-generation ethanol has been proposed. However, the microorganisms responsible for its production are not able to degrade structural polysaccharides, so their hydrolysis is necessary. Previously to this work, a factorial experimental design was carried out to investigate the relation between the NaOH and H₂O₂ concentrations with the yield of carbohydrates, and then this variable was optimized by using a response surface method. A study of the hydrolysis process was performed using enzymes to establish a process that maximizes the depolymerization of Agave tequilana fibers after an alkali-oxidative pretreatment with optimal reagents concentrations, this pretreatment was selected because it can remove almost the total content of lignin and destroys efficiently the crystallinity of cellulose fibers with a lower sugar losses and no production of toxic compounds. An orthogonal array using the novel enzymes Cellic CTec 3 and Cellic HTec 3 was performed to determinate the optimal combination of them, which has resulted in a concentration of 165.67 g/L at the supernatant with 82.21 % conversion and a yield of 352.18 g reducing sugars per kilogram of lignocellulosic material in dry basis. These results are 29 % better in comparison with the previous generation of enzymes with a reduction in the enzymatic charge of 82 %.     

Functional Approach to the Catalytic Site of the Sarcoplasmic Reticulum Ca2+-ATPase: Binding and Hydrolysis of ATP in the Absence of Ca2+

Isolated sarcoplasmic reticulum vesicles in the presence of Mg(2+) and absence of Ca(2+) retain significant ATP hydrolytic activity that can be attributed to the Ca(2+)-ATPase protein. At neutral pH and the presence of 5 mM Mg(2+), the dependence of the hydrolysis rate on a linear ATP concentration scale can be fitted by a single hyperbolic function. MgATP hydrolysis is inhibited by either free Mg(2+) or free ATP. The rate of ATP hydrolysis is not perturbed by vanadate, whereas the rate of p-nitrophenyl phosphate hydrolysis is not altered by a nonhydrolyzable ATP analog. ATP binding affinity at neutral pH and in a Ca(2+)-free medium is increased by Mg(2+) but decreased by vanadate when Mg(2+) is present. It is suggested that MgATP hydrolysis in the absence of Ca(2+) requires some optimal adjustment of the enzyme cytoplasmic domains. The Ca(2+)-independent activity is operative at basal levels of cytoplasmic Ca(2+) or when the Ca(2+) binding transition is impeded.     

Modeling technology innovation: How science, engineering, and industry methods can combine to generate beneficial socioeconomic impacts

Background

The introduction of evidence-based programs and practices into healthcare settings has been the subject of an increasing amount of research in recent years. While a number of studies have examined initial implementation efforts, less research has been conducted to determine what happens beyond that point. There is increasing recognition that the extent to which new programs are sustained is influenced by many different factors and that more needs to be known about just what these factors are and how they interact. To understand the current state of the research literature on sustainability, our team took stock of what is currently known in this area and identified areas in which further research would be particularly helpful. This paper reviews the methods that have been used, the types of outcomes that have been measured and reported, findings from studies that reported long-term implementation outcomes, and factors that have been identified as potential influences on the sustained use of new practices, programs, or interventions. We conclude with recommendations and considerations for future research.

Methods

Two coders identified 125 studies on sustainability that met eligibility criteria. An initial coding scheme was developed based on constructs identified in previous literature on implementation. Additional codes were generated deductively. Related constructs among factors were identified by consensus and collapsed under the general categories. Studies that described the extent to which programs or innovations were sustained were also categorized and summarized.

Results

Although "sustainability" was the term most commonly used in the literature to refer to what happened after initial implementation, not all the studies that were reviewed actually presented working definitions of the term. Most study designs were retrospective and naturalistic. Approximately half of the studies relied on self-reports to assess sustainability or elements that influence sustainability. Approximately half employed quantitative methodologies, and the remainder employed qualitative or mixed methodologies. Few studies that investigated sustainability outcomes employed rigorous methods of evaluation (e.g., objective evaluation, judgement of implementation quality or fidelity). Among those that did, a small number reported full sustainment or high fidelity. Very little research has examined the extent, nature, or impact of adaptations to the interventions or programs once implemented. Influences on sustainability included organizational context, capacity, processes, and factors related to the new program or practice themselves.

Conclusions

Clearer definitions and research that is guided by the conceptual literature on sustainability are critical to the development of the research in the area. Further efforts to characterize the phenomenon and the factors that influence it will enhance the quality of future research. Careful consideration must also be given to interactions among influences at multiple levels, as well as issues such as fidelity, modification, and changes in implementation over time. While prospective and experimental designs are needed, there is also an important role for qualitative research in efforts to understand the phenomenon, refine hypotheses, and develop strategies to promote sustainment.     

Modeling technology innovation: How science, engineering, and industry methods can combine to generate beneficial socioeconomic impacts

ABSTRACT: BACKGROUND: Government-sponsored science, technology, and innovation (STI) programs support the socioeconomic aspects of public policies, in addition to expanding the knowledge base. For example, beneficial healthcare services and devices are expected to result from investments in research and development (R&D) programs, which assume a causal link to commercial innovation. Such programs are increasingly held accountable for evidence of impact--that is, innovative goods and services resulting from R&D activity. However, the absence of comprehensive models and metrics skews evidence gathering toward bibliometrics about research outputs (published discoveries), with less focus on transfer metrics about development outputs (patented prototypes) and almost none on econometrics related to production outputs (commercial innovations). This disparity is particularly problematic for the expressed intent of such programs, as most measurable socioeconomic benefits result from the last category of outputs. METHODS: This paper proposes a conceptual framework integrating all three knowledge-generating methods into a logic model, useful for planning, obtaining, and measuring the intended beneficial impacts through the implementation of knowledge in practice. Additionally, the integration of the Context-Input-Process-Product (CIPP) model of evaluation proactively builds relevance into STI policies and programs while sustaining rigor. RESULTS: The resulting logic model framework explicitly traces the progress of knowledge from inputs, following it through the three knowledge-generating processes and their respective knowledge outputs (discovery, invention, innovation), as it generates the intended sociobeneficial impacts. It is a hybrid model for generating technology-based innovations, where best practices in new product development merge with a widely accepted knowledgetranslation approach. Given the emphasis on evidence-based practice in the medical and health fields and "bench to bedside" expectations for knowledge transfer, sponsors and grantees alike should find the model useful for planning, implementing, and evaluating innovation processes. CONCLUSIONS: High-cost/high-risk industries like healthcare require the market deployment of technologybased innovations to improve domestic society in a global economy. An appropriate balance of relevance and rigor in research, development, and production is crucial to optimize the return on public investment in such programs. The technology-innovation process needs a comprehensive operational model to effectively allocate public funds and thereby deliberately and systematically accomplish socioeconomic benefits.     

A Low-Km 5'-Nucleotidase from Rat Brain Cytosolic Fraction: Purification, Kinetic Properties, and Description of Regulation by a Novel Factor that Increases Sensitivity to Inhibition by ATP and ADP

Abstract: A readily soluble 5'-nucleotidase was purified 1,800-fold from rat brain 105,000- g supernatant. The enzyme showed similarity to the 5'-nucleotidase ectoenzyme of plasma membranes. It exhibited a low K _m for AMP, which was preferred over IMP as substrate. It was inhibited by free ATP and ADP and by α,β-methylene ADP. The enzyme appeared to be a glycoprotein on the basis of its interaction with concanavalin A. It contained a phosphatidylinositol moiety because treatment with phosphatidylinositol-specific phospholipase C increased its hydrophilicity. A single subunit of M_r = 54,300 ± 800 was observed, which is appreciably smaller than published values for the 5'-nucleotidase ectoenzyme or for other low- K _m"soluble" 5'-nucleotidases. The soluble 5'-nucleotidase showed an elution profile on AMP-Sepharose affinity chromatography or on Mono Q ion-exchange chromatography different from that of the brain ectoenzyme. Forty-two percent of the soluble 5'-nucleotidase in brain 105,000- g supernatant did not bind to a Mono Q ion-exchange column because of its interaction with a soluble factor. This factor could be removed by chromatography on concanavalin A-Sepharose. The factor had the novel property of increasing the sensitivity of the purified soluble 5'-nucleotidase toward the inhibitor ATP by 20-fold. This factor was also able to increase the inhibition of brain 5'-nucleotidase ectoenzyme by ATP.