Tissue-type plasminogen activator variants with domain duplications and rearrangements

The interactions between tPA domains that are important forcatalysis are poorly understood. We have probed the functionof interdomain interactions by generating tPA variants in whichdomains are duplicated or rearranged. The proteins were expressedin a transient mammalian expression system and tested in vitrofor their ability to activate plasminogen, induce fibrinolysisand bind to a forming fibrin clot. Duplication of the heavychain domains of tPA produced enzymatically active tPA variants,many of which demonstrated similar in vitro amidolytic and fibrinolyticactivity and similar fibrin affinity to the parent molecule.Zymographic analysis of the domain duplication tPA variantsshowed one major active species for each variant. Selectionof the residues duplicated and the interdomain spacing werefound to be critical considerations in the design of tPA variantswith duplicated domains. We also rearranged the domains of tPAsuch that kringle 1 replaced the second kringle domain and viceversa. An analysis of these variants indicates that the firstkringle domain can confer fibrin affinity to a tPA variant andfunction in place of kringle 2. Therefore, in wild-type tPA,the functions of kringle 1 and kringle 2 must be dependent partiallyon their orientation within the heavy chain of the protein.The functional autonomy of the heavy and light chains of tPAis demonstrated by the activity of a tPA variant in which theorder of the heavy and light chains was reversed.     

Iterative optimization of high-affinity proteases inhibitors using phage display. 1. Plasmin

We generated a series of libraries having variants of the first Kunitz domain of human lipoprotein-associated coagulation inhibitor (LACI-D1, also known as tissue-factor pathway inhibitor-I) displayed on bacteriophage M13 as pIII-fusions. We varied LACI-DI iteratively in two regions: the P1 region (positions 10-21) and the "second loop", (positions 31-39), which together form one end of the domain. Display-phage library Lib#1 allows 31 200 amino-acid sequences in P1 region (residues 13, 16-19). Preliminary, we screened Lib#1 against human plasmin (PLA, EC 3.4.21.7) immobolized on agarose to enrich for phage displaying variants with PLA affinity. We introduced a 1600-fold increase in second-loop diversity (residues 31, 32, 34, 39) into the population of selectants from Lib#1, yielding Lib#2. Lib#2 (allowing approximately 50 million amino-acid sequences) was screened against PLA-agarose to isolate highest affinity binders. Protein EPI-P211, derived from the best isolate of Lib#2, inhibits PLA with Ki = 2 nM (at least 500-fold better than LACI-D1) and with high specificity. We used amino-acid sequences of PLA-binding selectants to design a PLA-biased library (Lib#3) which we screened against PLA. The protein EPI-P302 (derived from the best binder obtained from Lib#3) has Ki for PLA inhibition of 87 pM, which is 25-fold better than the first-round best binder and > or = 12 500-fold better than LACI-D1. EPI-P302 also shows very high specificity for PLA vs other human proteases and is resistant to inactivation by oxidants and extremes of temperature or pH. Thus, one can use selectants from one library to design target-tailored combinatorial libraries and obtain quite stable, highly specific, very high-affinity binding molecules while maintaining an essentially human framework.     

Due Date Assignment in a Multistage Job Shop

In a Materials Requirements Planning (MRP) system, component lead times are generally assumed to be known and constant. Since a requirement of a MRP system is that components finish on time, slack is often built into component lead times to insure that actual job flowtimes equal planned job lead times. In this study, job characteristics and shop characteristics are investigated to determine which has a greater impact on predicting job flowtimes. This is accomplished by determining which characteristic most greatly influences the deviation of planned job lead times from actual job flowtimes. After identifying those factors which have the greatest influence on the job's flowtime, due date assignment rules are developed. A simulation study is then made to determine. which of the due date assignment rules perform best for varying product structures, as defined by various Bills of Materials (BOMs), and various shop conditions. Simulation test results are presented and discussed.     

Comparison of the single-dose pharmacokinetics and tolerability of modafinil and dextroamphetamine administered alone or in combination in healthy male volunteers

An open-label, randomized, crossover study was performed in healthy male volunteers to evaluate the potential pharmacokinetic and pharmacodynamic interactions and tolerability of single oral doses of modafinil (200 mg) and dextroamphetamine (10 mg). Blood samples were collected for determination of plasma levels of modafinil, the acid and sulfone metabolites of modafinil, and dextroamphetamine at intervals through 48 hours after administration for each treatment. Vital signs (blood pressure and pulse rate) were measured through 48 hours, and electrocardiograms were measured through 24 hours after administration. Pharmacokinetic parameters were determined using noncompartmental methods. The data collected in this study of 24 healthy volunteers suggest that concomitant administration of single oral doses of modafinil and dextroamphetamine has no clinically significant effects on the pharmacokinetic profile of either agent. Although there was a slightly greater incidence of adverse events when modafinil and dextroamphetamine were administered together, the concomitant administration of the two drugs was well tolerated.     

Analyzing commodity market trading strategies within a corporate environment

Improvement of the planning of commodity purchases remains a formidable problem in many agri-business firms. This paper addresses this problem within the corn purchasing environment of a large American food company. A time-horizon simulation model is developed for testing various commodity purchasing plans. The model is used to analyze interactions between various hedging strategies, profit-taking rules, and stop-loss rules, over an extended time period, under simulated market conditions. Extensive test results are presented and discussed.     

Numerical computation of optimal control

The formal solution for dynamically optimized control of a process is specified in terms of a two-point boundary problem. The numerical computation then presents great difficulties and speed of convergence is vital, even with the most powerful of contemporary computers. The authors give a method for rapid final convergence in which the two-point boundary problem is avoided altogether, although it is in fact only slightly different to methods of second variations. Computing experience with two examples is then described: a) aircraft landing and b) control of a boiler, with which the speed of convergence is well illustrated.     

Structure-function analysis of tissue-type plasminogen activator by linker-insertion, point and deletion mutagenesis

We undertook a structure–function analysis of human tissueplasminogen activator (tPA) using linker-scanning and deletionmutagenesis. Synthetic oligonucleotide linkers were introducedinto the tPA cDNA at pre-existing restriction enzyme sites.This generated a series of tPA variants which contained smallprimary sequence alterations consisting of point mutations,deletions or insertions. The majority of the linker-insertionvariants demonstrate a significant reduction in amidolytic andfibrinolytic activity in comparison to wild-type tPA. The exceptionsare the variants with linker-inserts placed at the BglII(115)and StyI(277) sites of the tPA cDNA (4SLEG5 and 57LEA58 respectively),which encode insertions at the boundaries of the finger domain.The variants with linker-inserts in the light chain (proteasedomain) of tPA are the lowest in enzymatic activity. Particularlysensitive to mutation are highly conserved amino acids. Heavychain deletion variants were constructed from point mutantsat the domain boundaries of tPA. Deletion of the kringle domainslowers the fibrinolytic activity to a greater extent than deletionof the finger or growth factor domains. We conclude that alterationsin any domain of the tPA molecule, and particularly in the highlyconserved residues within these domains, can affect fibrinolyticactivity.     

Self-disgust mediates the relationship between dysfunctional cognitions and depressive symptomatology.

Disgust has been linked to several psychopathologies, although a role in depression has been questioned. However, it has recently been proposed that rather than general disgust sensitivity, disgust directed toward the self (self-disgust) may influence the development of depression, providing a causal link between dysfunctional cognitions and depressive symptomatology. This possibility was examined by developing a scale to measure self-disgust (the Self-Disgust Scale; SDS) and then using mediator analysis to determine if self-disgust was able to explain the relationship between dysfunctional cognitions (measured with the use of the Dysfunctional Attitudes Scale) and depressive symptomatology (measured with the use of the Beck Depression Inventory and the Depression, Anxiety and Stress Scale). The developed SDS was found to exhibit a high level of internal consistency, test-retest reliability, and concurrent validity. Principal-components analysis revealed two factors to underlie responses to SDS items: the 'Disgusting self,' concerned with enduring, context independent aspects of the self, and 'Disgusting ways,' concerned with behavior. Self-disgust was found to mediate the relationship between dysfunctional cognitions and depressive symptomatology, demonstrating for the first time that self-disgust plays a role in depression. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Learning better IV&V practices

After data mining National Aeronautics and Space Administration (NASA) independent verification and validation (IV&V) data, we offer (a) an early life cycle predictor for project issue frequency and severity; (b) an IV&V task selector (that used the predictor to find the appropriate IV&V tasks); and (c) pruning heuristics describing what tasks to ignore, if the budget cannot accommodate all selected tasks. In ten-way cross-validation experiments, the predictor performs very well indeed: the average f-measure for predicting four classes of issue severity was over 0.9. This predictor is built using public-domain data and software. To the best of our knowledge, this is the first reproducible report of a predictor for issue frequency and severity that can be applied early in the life cycle. This research was conducted at West Virginia University and the NASA IV&V Facility under NASA subcontract project 100005549, task 5e, award 1002193r. Reference herein to any specific commercial product, process, or service by trade name, trademark, manufacturer, or otherwise, does not constitute or imply its endorsement by the United States Government. See for an earlier draft.     

Steroid hormone receptor studies in melanoma model systems

The transplantable B-16 melanotic melanoma carried in syngeneic C57Bl/6J female mice and the Syrian hamster melanoma cell line, RPMI 3460, were utilized to determine whether steroid-hormone receptors are present in animal melanomas. In the B-16 melanoma, a cytoplasmic-estrogen receptor is detectable, but there is no evidence for androgen or progestin receptors. Some tumors contain a glucocorticoid-binding macromolecule. Sucrose-density gradient centrifugation of cytosol after incubation with [3H]-estradiol revealed an 8S peak that was suppressed by excess radioinert diethylstilbesterol. Binding varied from 5-35 fmoles per mg cytosol protein. Scatchard analysis of [3H]-estradiol binding in cytosol yielded a single class of high-affinity binding sites; the dissociation constant is 6 x 10(-10) M. The receptor molecule is shown to be estrogen-specific by ligand competition assays. In contrast to B-16 melanoma, no estrogen, androgen, or progestin receptor can be found in the Syrian hamster melanoma cell line. However, a substantial level of specific binding is observed using [3H]-dexamethasone. Sucrose-gradient centrifugation of cytosol from this cell line after incubation with [3H]-dexamethasone revealed a 7S peak that was suppressed by excess radioinert dexamethasone. Scatchard analysis indicated a single class of high-affinity sites with a dissociation constant of 2 x 10(-9) M. Binding levels from 70-610 fmoles per mg cytosol protein were observed. The Syrian hamster melanoma cells also exhibit a biological response to glucocorticoids: Dexamethasone causes both an inhibition of growth and a decrease in final-cell density in these cells.