scholarly journals The identification of intermediates in the reaction of pig heart lactate dehydrogenase with its substrates

1974 ◽  
Vol 139 (3) ◽  
pp. 677-697 ◽  
Author(s):  
J. R. Whitaker ◽  
D. W. Yates ◽  
N. G. Bennett ◽  
J. J. Holbrook ◽  
H. Gutfreund
Keyword(s):  
Steady State ◽  
Lactate Dehydrogenase ◽  
Rate Constant ◽  
Active Sites ◽  
Phase 1 ◽  
Lactate Concentration ◽  
Phenol Red ◽  
Phase 2 ◽  
Protein Fluorescence ◽  
Rapid Formation

Pig heart lactate dehydrogenase was studied in the direction of pyruvate and NADH formation by recording rapid changes in extinction, proton concentration, nucleotide fluorescence and protein fluorescence. Experiments measuring extinction changes show that there is a very rapid formation of NADH within the first millisecond and that the amplitude of this phase (phase 1) increases threefold over the pH range 6–8. A second transient rate (phase 2) can also be distinguished (whose rate is pH-dependent), followed by a steady-state rate (phase 3) of NADH production. The sum of the amplitudes of the first two phases corresponds to 1mol of NADH produced/mol of active sites of lactate dehydrogenase. Experiments that measured the liberation of protons by using Phenol Red as an indicator show that no proton release occurs during the initial very rapid formation of NADH (phase 1), but protons are released during subsequent phases of NADH production. Fluorescence experiments help to characterize these phases, and show that the very rapid phase 1 corresponds to the establishment of an equilibrium between ENADLactate ⇌ H+ENADHPyruvate. This equilibrium can be altered by changing lactate concentration or pH, and the H+ENADHPyruvate species formed has very low nucleotide fluorescence and quenched protein fluorescence. Phase 2 corresponds to the dissociation of pyruvate and a proton from the complex with a rate constant of 1150s-1. The observed rate constant is slower than this and is proportional to the position of the preceding equilibrium. The ENADH formed has high nucleotide fluorescence and quenched protein fluorescence. The reaction, which is rate-limiting during steady-state turnover, must then follow this step and be involved with dissociation of NADH from the enzyme or some conformational change immediately preceding dissociation. Several inhibitory complexes have also been studied including ENAD+Oxamate and ENADHOxamate' and the abortive ternary complex ENADHLactate. The rate of NADH dissociation from the enzyme was measured and found to be the same whether measured by ligand displacement or by relaxation experiments. These results are discussed in relation to the overall mechanism of lactate dehydrogenase turnover and the independence of the four binding sites in the active tetramer.

scholarly journals A Population Pharmacokinetic Model for Concizumab Based on Phase 1 and Phase 2 Trial Data

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 4-4
Author(s):  
Trine Høyer Rose ◽  
Christian Hollensen ◽  
Henrik Agersø ◽  
Rune Viig Overgaard
Keyword(s):  
Rate Constant ◽  
Drug Disposition ◽  
Hemophilia A ◽  
Phase 1 ◽  
Population Pharmacokinetic ◽  
Phase 2 ◽  
Publicly Traded ◽  
Phase 3 ◽  
Target Mediated Drug Disposition ◽  
Population Pk

Introduction Concizumab is a high-affinity anti-tissue factor pathway inhibitor (TFPI) monoclonal antibody in clinical investigation for the subcutaneous (SC) treatment of patients with hemophilia. The data generated from phase 1 and 2 concizumab trials have been used to develop a population pharmacokinetic (PK) model with the aim of supporting dose selection for phase 3 trials. WMethods The objective of this study was to develop a model to describe the PK of concizumab across administration routes in various groups of patients with hemophilia to generate a generally applicable population PK model of concizumab. The model was developed based on available PK data from four phase 1 trials (for both intravenous [IV] and SC concizumab administration) and two phase 2 trials (for SC concizumab administration). Trial populations in the phase 1 trials included both healthy subjects and patients with hemophilia, whilst the phase 2 trials enrolled patients with hemophilia A or B with inhibitors and patients with hemophilia A without inhibitors. A structural population PK model was first developed based on phase 1 data and the final population PK model was then estimated using data from both phase 1 and phase 2 trials. Simulations were performed for phase 3 concizumab exposure using a full parametric simulation (n=10,000), including both inter-individual and intra-individual variability for the selected population. Randomly sampled body weights from a normal distribution with mean and variance corresponding to body weight distribution from phase 2 trials were used to simulate patient profiles. WResults The population PK dataset used for the model comprised 1,504 observations from 119 subjects (89 patients and 30 healthy individuals), with a mean age of 35 years (range: 18-65 years) and mean body weight of 74.4 kg (range: 47.1-130 kg). The PK model parameters were first estimated based on phase 1 data alone, and after fixing the majority in order to ensure robustness of the model only a few parameters were re-estimated based on phase 1 and 2 data combined. The PK model (Figure 1) was evaluated by standard goodness-of-fit plots and qualification assessments. Using visual predictive checks, it was shown that the model was able to reproduce the median and the 5th and 95th percentiles of the observed concizumab concentrations from phase 1 and 2 trials, and so it was deemed suitable for simulation purposes. The PK model suggested a target-mediated drug disposition following concizumab binding to TFPI at the endothelium, and subsequent elimination of the complex to account for the non-linear elimination. WConclusions The developed model accurately described the PK of concizumab delivered at a wide dose range by either SC or IV administration to both healthy subjects and patients with hemophilia A or B with and without inhibitors. The model was used for simulations to select the dosing regimen for subsequent phase 3 studies. Figure 1. Concizumab pharmacokinetic model. Structure of the final concizumab PK model for SC and IV dosing with target-mediated drug disposition via the endothelial TFPI. CL, clearance; doseiv, intravenous dose; dosesc, subcutaneous dose; IV, intravenous; ka, absorption rate constant; kcom, elimination rate constant of the concizumab-TFPI complex; kon and koff, rate constants for binding of concizumab to the endothelial TFPI; ktr, rate constant from the transit compartment; Q, inter-compartmental clearance; Rtot, amount of endothelial TFPI available for concizumab binding; SC, subcutaneous; TFPI, tissue factor pathway inhibitor; V, volume. Figure Disclosures Høyer Rose: Novo Nordisk A/S: Current Employment, Divested equity in a private or publicly-traded company in the past 24 months. Hollensen:Novo Nordisk: Current Employment, Current equity holder in private company, Current equity holder in publicly-traded company. Agersø:Novo Nordisk A/S: Current Employment. Viig Overgaard:Novo Nordisk A/S: Current Employment, Current equity holder in publicly-traded company.

Perception of Depth in Rotating Objects: 4. Fluctuating Stereokinetic Perceptual Variants

1968 ◽  
Vol 27 (1) ◽  
pp. 255-276 ◽  
Author(s):  
Roy B. Mefferd
Keyword(s):  
Steady State ◽  
Perceptual Organization ◽  
Phase 1 ◽  
Apparent Depth ◽  
Phase 2 ◽  
Basic System ◽  
Axis Of Rotation ◽  
Visual Systems ◽  
Processing Information ◽  
The One

There are a variety of stereokinetic percepts that fluctuate suddenly and unpredictably. On first viewing their sequence is predictable (Phase 1), but later it becomes unpredictable (Phase 2). A given percept results from the interplay of three independent visual systems: (1) the near-far mechanism (perspective reversals), (2) the one object-multiple object mechanism (degree of perceptual organization), and (3) the anchor point mechanism (apparent location of the axis of rotation). It was hypothesized that these fluctuating phenomena result from the operation of a basic system for repetitively sampling and pre-processing information on orientation, perceptual organization, and location of the axis of rotation and that a steady-state apparent-depth mechanism stabilizes the systems.

scholarly journals The kinetics of interconversion of intermediates of the reaction of pig muscle lactate dehydrogenase with oxidized nicotinamide–adenine dinucleotide and lactate

1973 ◽  
Vol 135 (1) ◽  
pp. 81-85 ◽  
Author(s):  
Nigel G. Bennett ◽  
Herbert Gutfreund
Keyword(s):  
Steady State ◽  
Lactate Dehydrogenase ◽  
Dead Time ◽  
Muscle Lactate ◽  
Rapid Formation ◽  
Steady State Rate ◽  
Pig Muscle ◽  
State Rate ◽  
Enzyme Complexes ◽  
Kinetics Of

Oxamate competes with pyruvate for the substrate binding site on the ENADH complex of pig skeletal muscle lactate dehydrogenase. When this enzyme was mixed with saturating concentrations of NAD+ and lactate in a stopped-flow rapid-reaction spectrophotometer there was no transient accumulation of enzyme complexes with the reduced nucleotide. The steady-state rate of formation of free NADH was reached within the dead-time of the instrument (3ms). When oxamate was added to inhibit the steady state and to uncouple the equilibration: [Formula: see text] through the rapid formation of ENADHOxamate, the rate of formation of ENADH could be measured by observation of the first turnover. This pH-dependent transient is controlled by the rate of dissociation of pyruvate and the fraction of the enzyme in the form ENADHPyruvate.

Phase II results of Study PX-171–007: A phase Ib/II study of carfilzomib (CFZ), a selective proteasome inhibitor, in patients with selected advanced metastatic solid tumors

2009 ◽  
Vol 27 (15_suppl) ◽  
pp. 3515-3515 ◽  
Author(s):  
P. J. Rosen ◽  
M. Gordon ◽  
P. N. Lee ◽  
E. Sausville ◽  
K. P. Papadopoulos ◽  
...  
Keyword(s):  
Solid Tumors ◽  
Proteasome Inhibitor ◽  
Active Sites ◽  
Phase 1 ◽  
Single Agent ◽  
Small Cell ◽  
Phase 2 ◽  
Small Cell Lung ◽  
Dose Escalation Study ◽  
C Cycle

3515 Background: CFZ is a novel proteasome inhibitor of the peptide epoxyketone class that exhibits a high level of selectivity for proteasome active sites. This phase 1/2 study assessed the maximum tolerated dose (MTD), safety, efficacy, pharmacokinetics (PK), and pharmacodynamics (PD) of CFZ in patients (pts) with advanced metastatic solid tumors. Methods: Pts failing ≥ 2 prior treatments were enrolled in the phase 1 3+3 dose escalation study. Pts received CFZ IV Day (D) 1, 2, 8, 9, 15 and 16 every 28 d for up to 12 cycles (C) Cycle 1 D1, D2 dosing in all cohorts was at 20 mg/m2. Subsequent doses remained at 20 mg/m2 or were escalated to 27 or 36 mg/m2 in a stepped up regimen on D8. At 20/36 mg/m2, 1 pt had a DLT (Grade 3 fatigue) and established the phase 2 dose. Phase 2 is designed as a Simon 2 stage of 70 pts split into 5 subgroups (small cell lung [SCLC], non-small cell lung [NSCLC], ovarian, renal, and other cancer). Tumor response was measured every 2 C. ORR, defined as CR+PR+SD, to 16 wks of CFZ. Stage 2 will open if a 1 PR or better at 16 wks occurs in a selected subgroup. Results: 14 pts in phase 1 and 51 pts in phase 2 (23M/28F, mean age 61 yrs) received a total of 154.5 cycles of CFZ. Median cycles administered was 1.7 (range 1 to 12). To date, there were 6 SCLC, 10 NSCLC, 11 ovarian, 6 renal, and 18 other cancer patients enrolled to the Simon stage 1. Efficacy of SD or better is detailed in the table. The most common AEs included fatigue headache, diarrhea, nausea and constipation. Notable was the absence of grade > 1 peripheral neuropathy and severe hematologic toxicities. Final results of the PK and PD will be reported. Conclusions: CFZ is active as a single agent in relapsed solid tumors demonstrating PR in both renal and SCLC; and SD >16 wks in mesothelioma, ovarian, renal and NSCLC. The 20/36 mg/m2 QDx2 dose schedule was well tolerated and lacks severe myelosuppression, hepatotoxicity and neuropathy which make CFZ an attractive agent to combine with traditional or novel targeted agents. [Table: see text] [Table: see text]

scholarly journals The kinetics of ox kidney biliverdin reductase in the pre-steady state. Evidence that the dissociation of bilirubin is the rate-determining step

1989 ◽  
Vol 259 (3) ◽  
pp. 709-713 ◽  
Author(s):  
E Rigney ◽  
T J Mantle ◽  
F M Dickinson
Keyword(s):  
Steady State ◽  
Rate Constant ◽  
Ph Dependence ◽  
Order Rate Constant ◽  
Protein Fluorescence ◽  
Biliverdin Reductase ◽  
Rate Determining Step ◽  
Steady State Rate ◽  
State Rate ◽  
Kinetics Of

When the production of bilirubin by biliverdin reductase was monitored at 460 nm by stopped-flow spectrophotometry a ‘burst’ was observed with a first-order rate constant at pH 8 of 20 s-1. The steady-state rate was established on completion of the ‘burst’. When the reaction was monitored at 401 nm there was no observed steady-state rate, but a diminished pre-steady-state ‘burst’ reaction was still seen with a rate constant of 22 s-1. We argue that the rate-limiting reaction is the dissociation of bilirubin from an enzyme.NADP+.bilirubin complex. With NADPH as the cofactor the hydride-transfer step was shown to exhibit pH-dependence associated with an ionizing group with a pK of 7.2. The kinetics of NADPH binding to the enzyme at pH 7.0 were measured by monitoring the quenching of protein fluorescence on binding the coenzyme.

scholarly journals Pre-steady-state kinetic studies on cytoplasmic sheep liver aldehyde dehydrogenase

1977 ◽  
Vol 167 (2) ◽  
pp. 469-477 ◽  
Author(s):  
Alastair K. H. MacGibbon ◽  
Leonard F. Blackwell ◽  
Paul D. Buckley
Keyword(s):  
Steady State ◽  
Isotope Effect ◽  
Aldehyde Dehydrogenase ◽  
Rate Constant ◽  
Kinetic Studies ◽  
Similar Data ◽  
Protein Fluorescence ◽  
Sheep Liver ◽  
Steady State Kinetic ◽  
Liver Aldehyde Dehydrogenase

Stopped-flow experiments in which sheep liver cytoplasmic aldehyde dehydrogenase (EC 1.2.1.3) was rapidly mixed with NAD+ and aldehyde showed a burst of NADH formation, followed by a slower steady-state turnover. The kinetic data obtained when the relative concentrations and orders of mixing of NAD+ and propionaldehyde with the enzyme were varied were fitted to the following mechanism: [Formula: see text] where the release of NADH is slow. By monitoring the quenching of protein fluorescence on the binding of NAD+, estimates of 2×105 litre·mol−1·s−1 and 2s−1 were obtained for k+1 and k−1 respectively. Although k+3 could be determined from the dependence of the burst rate constant on the concentration of propionaldehyde to be 11s−1, k+2 and k−2 could not be determined uniquely, but could be related by the equation: (k−2+k+3)/k+2 =50×10−6mol·litre−1. No significant isotope effect was observed when [1-2H]propionaldehyde was used as substrate. The burst rate constant was pH-dependent, with the greatest rate constants occurring at high pH. Similar data were obtained by using acetaldehyde, where for this substrate (k−2+k+3)/k+2=2.3×10 −3mol·litre−1 and k+3 is 23s−1. When [1,2,2,2-2H]acetaldehyde was used, no isotope effect was observed on k+3, but there was a significant effect on k+2 and k−2. A burst of NADH production has also been observed with furfuraldehyde, trans-4-(NN-dimethylamino)cinnamaldehyde, formaldehyde, benzaldehyde, 4-(imidazol-2-ylazo)benzaldehyde, p-methoxybenzaldehyde and p-methylbenzaldehyde as substrates, but not with p-nitrobenzaldehyde.

Rationalization and internalization

2001 ◽  
Vol 60 (4) ◽  
pp. 215-230 ◽  
Author(s):  
Jean-Léon Beauvois
Keyword(s):  
Phase 1 ◽  
Facilitatory Effect ◽  
Phase 2 ◽  
Causal Explanations ◽  
Reduction Effect ◽  
Dissonance Reduction

After having been told they were free to accept or refuse, pupils aged 6–7 and 10–11 (tested individually) were led to agree to taste a soup that looked disgusting (phase 1: initial counter-motivational obligation). Before tasting the soup, they had to state what they thought about it. A week later, they were asked whether they wanted to try out some new needles that had supposedly been invented to make vaccinations less painful. Agreement or refusal to try was noted, along with the size of the needle chosen in case of agreement (phase 2: act generalization). The main findings included (1) a strong dissonance reduction effect in phase 1, especially for the younger children (rationalization), (2) a generalization effect in phase 2 (foot-in-the-door effect), and (3) a facilitatory effect on generalization of internal causal explanations about the initial agreement. The results are discussed in relation to the distinction between rationalization and internalization.

PENERAPAN MODEL QUANTUM LEARNING UNTUK MENINGKATKAN HASIL BELAJAR AUTOCAD SISWA KELAS X TEKNIK PEMESINAN SMK NEGERI 1 STABAT

2013 ◽  
Vol 5 (1) ◽  
Author(s):  
Abdul Hasan Saragih
Keyword(s):  
Positive Response ◽  
Phase 1 ◽  
First Grade ◽  
Learning Model ◽  
Second Phase ◽  
Phase 2 ◽  
Phase 3 ◽  
The Third ◽  
Third Phase ◽  
Quantum Learning

This classroom research was conducted on the autocad instructions to the first grade of mechinary class of SMK Negeri 1 Stabat aiming at : (1) improving the student’ archievementon autocad instructional to the student of mechinary architecture class of SMK Negeri 1 Stabat, (2) applying Quantum Learning Model to the students of mechinary class of SMK Negeri 1 Stabat, arising the positive response to autocad subject by applying Quantum Learning Model of the students of mechinary class of SMK Negeri 1 Stabat. The result shows that (1) by applying quantum learning model, the students’ achievement improves significantly. The improvement ofthe achievement of the 34 students is very satisfactory; on the first phase, 27 students passed (70.59%), 10 students failed (29.41%). On the second phase 27 students (79.41%) passed and 7 students (20.59%) failed. On the third phase 30 students (88.24%) passed and 4 students (11.76%) failed. The application of quantum learning model in SMK Negeri 1 Stabat proved satisfying. This was visible from the activeness of the students from phase 1 to 3. The activeness average of the students was 74.31% on phase 1,81.35% on phase 2, and 83.63% on phase 3. (3) The application of the quantum learning model on teaching autocad was very positively welcome by the students of mechinary class of SMK Negeri 1 Stabat. On phase 1 the improvement was 81.53% . It improved to 86.15% on phase 3. Therefore, The improvement ofstudent’ response can be categorized good.

In situ deteriorating patient simulation in general practice

2020 ◽  
Vol 70 (suppl 1) ◽  
pp. bjgp20X711425
Author(s):  
Joanna Lawrence ◽  
Petronelle Eastwick-Field ◽  
Anne Maloney ◽  
Helen Higham
Keyword(s):  
Life Support ◽  
Early Recognition ◽  
Phase 1 ◽  
Patient Simulation ◽  
Medical Emergency ◽  
Phase 2 ◽  
Action Plans ◽  
Integrated Simulation ◽  
Deteriorating Patient

BackgroundGP practices have limited access to medical emergency training and basic life support is often taught out of context as a skills-based event.AimTo develop and evaluate a whole team integrated simulation-based education, to enhance learning, change behaviours and provide safer care.MethodPhase 1: 10 practices piloted a 3-hour programme delivering 40 minutes BLS and AED skills and 2-hour deteriorating patient simulation. Three scenarios where developed: adult chest pain, child anaphylaxis and baby bronchiolitis. An adult simulation patient and relative were used and a child and baby manikin. Two facilitators trained in coaching and debriefing used the 3D debriefing model. Phase 2: 12 new practices undertook identical training derived from Phase 1, with pre- and post-course questionnaires. Teams were scored on: team working, communication, early recognition and systematic approach. The team developed action plans derived from their learning to inform future response. Ten of the 12 practices from Phase 2 received an emergency drill within 6 months of the original session. Three to four members of the whole team integrated training, attended the drill, but were unaware of the nature of the scenario before. Scoring was repeated and action plans were revisited to determine behaviour changes.ResultsEvery emergency drill demonstrated improved scoring in skills and behaviour.ConclusionA combination of: in situ GP simulation, appropriately qualified facilitators in simulation and debriefing, and action plans developed by the whole team suggests safer care for patients experiencing a medical emergency.

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