Identification Of Rate-limiting Step Research Articles

Draft genome sequence and detailed characterization of biofuel production by oleaginous microalga Scenedesmus quadricauda LWG002611

BackgroundDue to scarcity of fossil fuel, the importance of alternative energy sources is ever increasing. The oleaginous microalgae have demonstrated their potential as an alternative source of energy, but have not achieved commercialization owing to some biological and technical inefficiency. Modern methods of recombinant strain development for improved efficacy are suffering due to inadequate knowledge of genome and limited molecular tools available for their manipulation.ResultsIn the present study, microalga Scenedesmus quadricauda LWG002611 was selected as the preferred organism for lipid production as it contained high biomass (0.37 g L−1 day−1) and lipid (102 mg L−1 day−1), compared to other oleaginous algae examined in the present study as well as earlier reports. It possessed suitable biodiesel properties as per the range defined by the European biodiesel standard EN14214 and petro-diesel standard EN590:2013. To investigate the potential of S. quadricauda LWG002611 in details, the genome of the organism was assembled and annotated. This was the first genome sequencing and assembly of S. quadricauda, which predicted a genome size of 65.35 Mb with 13,514 genes identified by de novo and 16,739 genes identified by reference guided annotation. Comparative genomics revealed that it belongs to class Chlorophyceae and order Sphaeropleales. Further, small subunit ribosomal RNA gene (18S rRNA) sequencing was carried out to confirm its molecular identification. S. quadricauda LWG002611 exhibited higher number of genes related to major activities compared to other potential algae reported earlier with a total of 283 genes identified in lipid metabolism. Metabolic pathways were reconstructed and multiple gene homologs responsible for carbon fixation and triacylglycerol (TAG) biosynthesis pathway were identified to further improve this potential algal strain for biofuel production by metabolic engineering approaches.ConclusionHere we present the first draft genome sequence, genetic characterization and comparative evaluation of S. quadricauda LWG002611 which exhibit high biomass as well as high lipid productivity. The knowledge of genome sequence, reconstructed metabolic pathways and identification of rate-limiting steps in TAG biosynthesis pathway will strengthen the development of molecular tools towards further improving this potentially one of the major algal strains for biofuel production.

Identification of Rate-Limiting Steps in the Provision of Thrombolytics for Acute Ischemic Stroke

Background: Tissue plasminogen activator (tPA) is the only pharmacotherapy shown to improve outcomes in acute ischemic stroke. The American Heart Association (AHA) recommends a door-to-needle (DTN) time of <60 minutes in at least 50% of patients presenting with acute ischemic stroke. Objective: The purpose of this study was to analyze the possible barriers that may delay tPA administration within the emergency department (ED) of an academic medical center. Methods: A retrospective chart review was conducted from February 2011 to October 2013. Patients were included if they were admitted through the ED with a diagnosis of acute ischemic stroke and received tPA. Results: Of the 130 patients who met inclusion criteria, 43.1% received tPA in ≤60 minutes. Several factors were identified to be significantly different in those with a DTN time of >60 minutes—time to ED physician consultation, neurologist arrival, blood sample acquisition, and result time (P < .05 for all comparisons). Correlation analysis demonstrated several independent variables associated with DTN time of ≤60 minutes—time from admission to ED physician consultation, receipt of computed tomography (CT) scan, blood sample acquisition, laboratory results, and neurology service arrival (P < .05 for all comparisons). Conclusion: The findings from this study highlight the importance of prompt physician evaluation, direct transfer to the CT scanner, and a quick turnaround time on laboratory values. The development of protocols to ensure the rapid receipt of tPA therapy should focus on limiting any potential delay these steps may cause.

Effect of pH on the metabolic flux of Klebsiella oxytoca producing 2,3-butanediol in continuous cultures at different dilution rates

The efficiency of the bioconversion process and the achievable end-product concentration decides the economic feasibility of microbial 2,3-butanediol (2,3-BDO) production. In 2,3-BDO production, optimization of culture condition is required for cell growth and metabolism. Also, the pH is an important factor that influences microbial performance. For different microorganisms and substrates, it has been shown that the distribution of the metabolites in 2,3-BDO fermentation is greatly affected by pH, and the optimum pH for 2,3-BDO production seems dependently linked to the particular strain and the substrate employed. Quantification analysis of intracellular metabolites and metabolic flux analysis (MFA) were used to investigate the effect of pH on the Klebsiella oxytoca producing 2,3-BDO and other organic acids. The main objectives of MFA are the estimation of intracellular metabolic fluxes and the identification of rate-limiting step and the key enzymes. This study was conducted under continuous aerobic conditions at different dilution rates (0.1, 0.2, and 0.3 h(-1)) and different pH values (pH 5.5 and 7.0) for the steady-state experimental data. In order to obtain the flux distribution, the extracellular specific rates were calculated from the experimental data using the metabolic network model of K. oxytoca. Intracellular metabolite concentration profiles were generated using ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry.

Enzymology under global change: organic nitrogen turnover in alpine and sub-Arctic soils

Understanding global change impacts on the globally important carbon storage in alpine, Arctic and sub-Arctic soils requires knowledge of the mechanisms underlying the balance between plant primary productivity and decomposition. Given that nitrogen availability limits both processes, understanding the response of the soil nitrogen cycle to shifts in temperature and other global change factors is crucial for predicting the fate of cold biome carbon stores. Measurements of soil enzyme activities at different positions of the nitrogen cycling network are an important tool for this purpose. We review a selection of studies that provide data on potential enzyme activities across natural, seasonal and experimental gradients in cold biomes. Responses of enzyme activities to increased nitrogen availability and temperature are diverse and seasonal dynamics are often larger than differences due to experimental treatments, suggesting that enzyme expression is regulated by a combination of interacting factors reflecting both nutrient supply and demand. The extrapolation from potential enzyme activities to prediction of elemental nitrogen fluxes under field conditions remains challenging. Progress in molecular '-omics' approaches may eventually facilitate deeper understanding of the links between soil microbial community structure and biogeochemical fluxes. In the meantime, accounting for effects of the soil spatial structure and in situ variations in pH and temperature, better mapping of the network of enzymatic processes and the identification of rate-limiting steps under different conditions should advance our ability to predict nitrogen fluxes.

The biosynthesis of the selectin-ligand sialyl Lewis x in colorectal cancer tissues is regulated by fucosyltransferase VI and can be inhibited by an RNA interference-based approach

Sialyl Lewis x (sLex) is a selectin ligand whose overexpression in epithelial cancers mediates metastasis formation. The molecular basis of sLex biosynthesis in colon cancer tissues is still unclear. The prerequisite for therapeutic approaches aimed at sLex down-regulation in cancer, is the identification of rate-limiting steps in its biosynthesis. We have studied the role of α1,3-fucosyltransferases (Fuc-Ts) potentially involved in sLex biosynthesis in specimens of normal and cancer colon as well as in experimental systems. We found that: (i) in colon cancer, but not in normal mucosa where the antigen was poorly expressed, sLex correlated with a Fuc-T which, like Fuc-TVI, was active on 3′sialyllactosamine at a low concentration (Fuc-T SLN); (ii) competitive RT-PCR analysis revealed that the level of Fuc-T mRNA expression in both normal and cancer colon was Fuc-TVI > Fuc-TIII > Fuc-TIV; Fuc-TV and Fuc-TVII expression was negligible; (iii) sLex was expressed only by the gastrointestinal cell lines displaying both Fuc-TVI mRNA and Fuc-T SLN activity, but not by those expressing only Fuc-TIII mRNA; (iv) transfection with Fuc-TVI cDNA, but not with Fuc-TIII cDNA, induced sLex expression in gastrointestinal cell lines; (v) Fuc-TVI knock-down with specific siRNA induced down-regulation of Fuc-TVI mRNA and Fuc-T SLN activity and a dramatic inhibition of sLex expression. These data indicate that in colon cancer tissues Fuc-TVI is a key regulator of sLex biosynthesis which can be the target of RNA-interference-based gene knock-down approaches.

Identification of rate-limiting steps in yeast heme biosynthesis

The heme biosynthesis pathway in the yeast Saccharomyces cerevisiae is a highly regulated system, but the mechanisms accounting for this regulation remain unknown. In an attempt to identify rate-limiting steps in heme synthesis, which may constitute potential regulatory points, we constructed yeast strains overproducing two enzymes of the pathway: the porphobilinogen synthase (PBG-S) and deaminase (PBG-D). Biochemical analysis of the enzyme-overproducing strains revealed intracellular porphobilinogen and porphyrin accumulation. These results indicate that both enzymes play a rate-limiting role in yeast heme biosynthesis.

Critical pathways : a review. Committee on Acute Cardiac Care, Council on Clinical Cardiology, American Heart Association.

Critical pathways, also known as critical paths, clinical pathways, or care paths, are management plans that display goals for patients and provide the sequence and timing of actions necessary to achieve these goals with optimal efficiency.1 As competition in the healthcare industry has increased, managers have embraced critical pathways as a method to reduce variation in care, decrease resource utilization, and potentially improve healthcare quality. Cardiovascular medicine in particular is an area in which critical pathways have been embraced. This is due in part to the high volume and high cost associated with cardiovascular diseases and procedures. In addition, the relatively mature guideline process has also contributed to the growth in use of critical pathways in cardiology. Although anchored in clinical guidelines, the critical pathway is a distinct tool that details processes of care and highlights inefficiencies regardless of whether there is evidence to warrant changes in those processes. Clinical guidelines, on the other hand, are consensus statements that are systematically developed to assist practitioners in making patient management decisions related to specific clinical circumstances.2 Although clinical guidelines can and should be used in pathway development, the majority of processes included in a pathway have not been rigorously tested and are generally not addressed in guidelines. Another term that should also be distinguished from critical pathways is clinical protocols. Protocols are treatment recommendations that are often based on guidelines. Like the critical pathway, the goal of the clinical protocol may be to decrease treatment variation. However, protocols are most often focused on guideline compliance rather than the identification of rate-limiting steps in the patient care process. In further contrast to critical pathways, protocols may or may not include a continuous monitoring and data-evaluation component. Critical pathway techniques were first developed for use in industry as a tool to …

Kinetics of insulin action in vivo. Identification of rate-limiting steps

Kinetics of insulin action in vivo. Identification of rate-limiting steps

Alcohol esterification reactions and mechanisms of snake venom 5'-nucleotide phosphodiesterase.

In a previous study we have shown that snake venom 5'-nucleotide phosphodiesterase (SVP) catalyzes methanol-esterification reactions [García-Díaz, M., Avalos, M. & Cameselle, J. C. (1991) Eur. J. Biochem. 196, 451-457]. Now we have demonstrated that SVP catalyzes AMP transfer from ATP to propanol, ethanol, methanol, ethylene glycol, glycerol, 2-chloroethanol or 2,2-dichloroethanol. The AMP-O-alkyl ester products were identified by HPLC, enzyme analysis, ultraviolet and NMR spectroscopy. Those results show the potential of SVP as a tool to prepare 5'-nucleotide esters and agree with the formation of a covalent 5'-nucleotidyl-SVP intermediate susceptible to nucleophilic attack by short-chain (poly)alcohols as acceptors alternative to water. To test the kinetic influence of the solvent nucleophile in SVP mechanisms, initial rates of ATP solvolysis were assayed in different water/alcohol mixtures. Relatively high alcohol concentrations inactivated SVP but lower concentrations gave proportional rates of alcoholysis. An efficiency parameter (EA), defined as the ratio of the mole fraction of AMP-O-alkyl ester as a product to that of alcohol as an acceptor in water/alcohol mixtures, made possible the comparison of alcohols and water as AMP acceptors at low concentrations, as it could be reasoned that EA = 1 for water. Rates of hydrolysis (VH) of substrates yielding AMP and different leaving groups were also assayed. The higher EA and VH values corresponded, respectively, to those acceptors and leaving-group conjugate acids with lower pKa and higher polar-substituent constants (sigma*). The results support the occurrence of general acid-base catalysis in the active center of SVP and the identification of rate-limiting steps. A model is proposed for the mechanisms of SVP-catalyzed hydrolysis and alcoholysis which accounts for the influence of the acid-base properties of alcohols on the kinetic profile of SVP reaction sequences.

Identification of rate-limiting steps in cephalosporin C biosynthesis in Cephalosporium acremonium: a theoretical analysis.

A kinetic model describing the biosynthesis of cephalosporin C in Cephalosporium acremonium has been developed to identify the rate-limiting step(s). Using this model and in-vitro kinetic data of the biosynthetic enzymes, the production kinetics of cephalosporin C were examined theoretically. The predicted time profile of the specific production rate during batch culture is in good agreement with that of experimental results published previously. Sensitivity analysis indicates that delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV) synthetase is the rate-limiting enzyme. Our analysis also predicts that increasing ACV synthetase enhances the production rate initially until expandase/hydroxylase becomes rate-limiting. Furthermore, increasing expandase/hydroxylase reduces the accumulation of penicillin N, and thus, enhances the production of cephalosporin C. Based on our analysis, amplifying both ACV synthetase and expandase/hydroxylase concurrently should enhance the production rate to a great extent.

Acetyl-coenzyme A synthesis from methyltetrahydrofolate, CO, and coenzyme A by enzymes purified from Clostridium thermoaceticum: attainment of in vivo rates and identification of rate-limiting steps.

Many anaerobic bacteria fix CO2 via the acetyl-coenzyme A (CoA) (Wood) pathway. Carbon monoxide dehydrogenase (CODH), a corrinoid/iron-sulfur protein (C/Fe-SP), methyltransferase (MeTr), and an electron transfer protein such as ferredoxin II play pivotal roles in the conversion of methyltetrahydrofolate (CH3-H4folate), CO, and CoA to acetyl-CoA. In the study reported here, our goals were (i) to optimize the method for determining the activity of the synthesis of acetyl-CoA, (ii) to evaluate how closely the rate of synthesis of acetyl-CoA by purified enzymes approaches the rate at which whole cells synthesize acetate, and (iii) to determine which steps limit the rate of acetyl-CoA synthesis. In this study, CODH, MeTr, C/Fe-SP, and ferredoxin were purified from Clostridium thermoaceticum to apparent homogeneity. We optimized conditions for studying the synthesis of acetyl-CoA and found that when the reaction is dependent upon MeTr, the rate is 5.3 mumol min-1 mg-1 of MeTr. This rate is approximately 10-fold higher than that reported previously and is as fast as that predicted on the basis of the rate of in vivo acetate synthesis. When the reaction is dependent upon CODH, the rate of acetyl-CoA synthesis is approximately 0.82 mumol min-1 mg-1, approximately 10-fold higher than that observed previously; however, it is still lower than the rate of in vivo acetate synthesis. It appears that at least two steps in the overall synthesis of acetyl-CoA from CH3-H4folate, CO, and CoA can be partially rate limiting. At optimal conditions of low pH (approximately 5.8) and low ionic strength, the rate-limiting step involves methylation of CODH by the methylated C/Fe-SP. At higher pH values and/or higher ionic strength, transfer of the methyl group of CH3-H4folate to the C/Fe-SP becomes rate limiting.

Resolution of gas phase and surface combustion chemistry into elementary reactions

It is well known for many decades, that combustion chemistry is a complex phenomenon, and realistic reaction mechanisms for simple hydrocarbons have been published in the last decade. Nevertheless, for 2D-or 3D-applications in combustion science and technology nearly exclusively one-step approaches are used at the present to make solutions possible within realistic computing times or to avoid principal physical problems (e.g. closure problems in modeling turbulent combustion). However, this is paid with a huge disadvantage: No extrapolation is possible to other experimental or operational conditions, and even interpolation is dangerous. This review describes how detailed reaction mechanisms can be developed for simple hydrocarbons and be verified using literature data on flame structure, flame propagation, and ignition from the literature; how these mechanisms can be extended to the treatment of very large hydrocarbons by use of additivity rules and processing of these rules by computer programs generating reaction mechanisms; how these large reaction mechanisms can be simplified, for use in multi-dimensional codes, e.g. by use of lumping techniques or by equilibration of fast reactions in a systematic way; how treatment in terms of sets of elementary reactions can be transferred to the handling of surface reactions, e.g. for the description of catalytic combustion. Examples are given for each of these developments. The special role of sensitivity analysis is emphasized in the understanding of detailed reaction mechanisms and the identification of rate-limiting steps which should be objects of further research efforts.

A comparison of energy-yielding reactions in the flight muscle of young adult and senescent blowflies

1. 1. Steady-state concentrations of key glycolytic intermediates and other metabolites were measured at rest and in flight muscle of young adult blowflies ( Phormia regina) and senescent blowflies fed either sugar and water (SW) or sugar, water and milk (SMW). 2. 2. This allowed the identification of rate-limiting steps in, these metabolic pathways and suggested changes at the level of trehalase, triosephosphate dehydrogenase and pyruvate kinase in the senescent SW fly and no change in the senescent SMW fly, compared to the young adult. 3. 3. Small decreases in triosephosphate dehydrogenase and pyruvate kinase activities could be directly demonstrated in enzyme assays of muscle extracts. 4. 4. No age-related changes in mitochondrial function could be demonstrated.