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Phytochrome Control of Specific mRNA levels in Developing Pea Buds : Kinetics of Accumulation, Reciprocity, and Escape Kinetics of the Low Fluence Response.

We have examined the time course for accumulation of each of 12 different nuclear gene transcripts in pea buds after irradiating dark grown seedlings with a single pulse low fluence red light (10(3) micromoles per square meter delivered in 100 seconds). The 12 time courses can be grouped into four general classes. Six transcripts (including RNAs coding for the chlorophyll a/b binding protein and ribulose-1,5-bisphosphate carboxylase) accumulate at a linear rate during 24 hours in darkness following the light pulse. Two transcripts increase rapidly at first but then reach a plateau after 3 hours and remain at that level for the next 21 hours. Another two transcripts exhibit a prolonged lag period before beginning to accumulate, and do not reach significant accumulation rates until 12 to 16 hours after the red light pulse. One transcript appears to undergo a transient increase in abundance in response to red light, but this is superimposed on a background of slowly increasing abundance of this RNA in control plants. This response, unlike all the others, exhibits reciprocity failure in experiments in which the same fluence of light is given over periods ranging between 50 and 4000 seconds.We have also examined the kinetics with which each of these 12 responses escapes from phytochrome-far-red absorbing form control by attempting to reverse the induction with far-red light given at various times after the red light pulse. Again, several different patterns are apparent for the different transcripts. The time at which far red reversibility first begins to be lost, the rate at which it is lost, and the final extent of reversibility remaining after 7 hours in the dark all differ for different transcripts. In addition, we have observed that some responses retain virtually complete photoreversibility for at least 7 hours. In some cases, a comparison of the time course and escape kinetic data indicates that relatively rapid turnover of the RNA must occur. It is not clear whether or not the rate of turnover is influenced by phytochrome.

Journal Article↗

Kinetics of the human medulloblastoma cell line, D283 Med, maintained in an organ culture system. Autoradiographic study after cumulative labelling in vitro: similarities to the kinetics of human medulloblastoma in situ.

Cells of the human D283 Med medulloblastoma cell line were collected by gravity sedimentation and maintained in vitro in an organ culture system. The explants grew as clusters of small cells with scanty cytoplasm; some cells were arranged in solid trabeculae, sometimes forming rows and columns, and occasionally rosettes. The kinetic parameters were estimated after 6 days in vitro by cumulative labelling of the cultures with tritiated thymidine in three different experiments. The length of G2 phase (TG2) was obtained from an analysis of the fractions of continuously labelled mitoses (FCLM), the length of S phase (TS) from the median values of grains over mitotic figures (grain index, GI), and the other parameters from mathematical models that related growth fraction (GF), TG2, total cell cycle time (TC) and labelling index (LI) as a function of time. Mean GF, TC, TG1, TS and TG2 were estimated respectively to be 0.35, 30 h, 13 h, 10 h, and 7 h. These estimates correspond to those reported for human medulloblastomas in situ and confirm previous in vitro experiments which suggest that the organ culture technique is a suitable system for the study of cell kinetics in human malignant gliomas and for an analysis of the in vitro effects of chemotherapeutic agents.

Autoradiography↗

Gender differences in labetalol kinetics: importance of determining stereoisomer kinetics for racemic drugs.

STUDY OBJECTIVE: To evaluate the impact of gender on labetalol kinetics. DESIGN: Part of a randomized, crossover study. SETTING: Academic medical center. PATIENTS: Nineteen hypertensive patients (14 men, 5 women; 6 blacks, 13 whites). INTERVENTIONS: Participants had labetalol dosages titrated to a specific antihypertensive response, then underwent ambulatory blood pressure monitoring (ABPM) and a pharmacokinetic study. Labetalol plasma concentrations were measured by high-performance liquid chromatography (HPLC) and labetalol stereoisomer ratios were determined in a single plasma sample by chiral HPLC, both with fluorescence detection. MEASUREMENTS AND MAIN RESULTS: Labetalol concentrations were 80% higher in women (area under the concentration-time curve [AUC]/dose x 1000: 6.79 +/- 2.11 in women vs 3.82 +/- 1.37 hr/L in men, p<0.05), yet both genders had a similar antihypertensive response by 24-hour ABPM. Dose-corrected AUC (AUC/dose x 1000) for labetalol's stereoisomers in women and men, respectively, were S,R-labetalol 7.55 +/- 1.47 and 4.83 +/- 1.54 hr/L (p<0.05), S,S-labetalol 8.23 +/- 2.93 and 4.65 +/- 1.78 hr/L (p<0.05), R,S-labetalol 6.99 +/- 3.30 and 4.25 +/- 2.35 hr/L (p=0.11), and R,R-labetalol 3.91 +/- 2.57 and 3.55 +/- 3.08 hr/L (NS). CONCLUSION: The higher labetalol concentration in women than in men was explained largely by differences in inactive and alpha1-blocking stereoisomers. However, concentrations were similar between genders for the beta-blocking stereoisomer (R,R-labetalol), possibly explaining the similarity in antihypertensive response to the drug. This study highlights the importance of determining stereoisomer kinetics for agents administered as racemates, particularly when relating concentrations to pharmacologic response.

Adult↗

[Kinetics of vaccination antibodies against tetanus toxoid, diphtheria toxoid, measles virus, poliomyelitis virus and pneumococcus after allogenic and autologous bone marrow transplantation and booster vaccination. 2: Kinetics of vaccination antibodies against diphtheria toxoid after allogenic and autologous bone marrow transplantation].

In the second part of the paper we report on the results of the diphtheria antitoxin valuation of 8 children after allogeneic bone marrow transplantation (BMT) with and without graft versus host disease (GvHD) as well as on the kinetics of the diphtheria antitoxin of 5 children after allogeneic BMT with and without GvHD and of 5 children after autologous transplantation. The antibody valuation was done by a cell-culture assay. Whereas the inspection of the isolated data gives the impression of a swift antibody decrease up to the non-protective level from the 7th month after BMT, the kinetic tests are more highly differentiated. Besides rapidly decreasing values below the accepted protection rate of 0.01 IU/ml in the allogeneic transplanted groups, there are patients with positive antibody titres within an observation period of up to 24 months after BMT in the allogeneic as well as in the autologous transplantation group.

Bone Marrow Transplantation↗

[Kinetics of vaccine antibodies to tetanus toxoid, diphtheria toxoid, measles virus, poliomyelitis virus and pneumococci after allogenic and autologous bone marrow transplantation and booster immunization. 1: The kinetics of vaccine antibodies to tetanus toxoid after allogenic and autologous bone marrow transplantation].

Today BMT belongs to the established methods of treatment in haematology and oncology. Because of the constant increase of healthy long-term survivors after BMT the problem of immunological reconstitution and eventual possible late effects gets more and more importance. One problem, which til now has been few attention paid to, is that of the protection by vaccination after BMT. We report on the kinetics of the tetanus-antitoxin in 20 patients after allogeneic or autologous BMT and demonstrate the influence of a graft-versus-host disease and its therapy on the antibody kinetics. In the group of allogeneic transplanted children without a GvHD the tetanus-antitoxin titers felt below their detection range after a time of about 8 months whereas in the group with GvHD this effect already occurred after nearly 4 months. The autologous transplanted patients have a positive antibody level til the time of 20 months after BMT. As a consequence of the lost protection by vaccination after BMT follows the necessity of revaccinations respectively of boostering after immunological reconstitution.

Adolescent↗

Kinetic description of proteolysis. Part 3. Total kinetics of peptide bonds hydrolysis in peptide mixtures.

A theoretical analysis of the general kinetics of peptide bond hydrolysis in peptide mixtures has been carried out. It is shown that the equation for the hydrolysis rate must include ensemble-averaged values which change as functions during hydrolysis. Thus the problem of describing the hydrolysis is reduced to finding these functions. As an example of theoretical prediction for the behavior of the averaged functions the procedure of calculating the averaged constant of chymotrypsin substrate specificity as a function of the degree of hydrolysis is presented. The analysis performed enables to find factors responsible for the decrease of hydrolysis rate, in particular, the role of the substrate specificity range and the S/E ratio. The design of specific experiments making it possible to measure the averaged values in the course of hydrolysis has been considered.

Chymotrypsin↗

Kinetic description of proteolysis. Part 4. Hydrolysis kinetics of partial protein hydrolysates.

This report presents experimental data on kinetics of casein hydrolysate hydrolysis. The experiments were interpreted in the framework of a theory reported, which is guided by an experimentally measurable value--a total molar concentration of all peptides and amino acids (total amino nitrogen). The total amino nitrogen N was determined by photometry of N-trinitrophenyl (N-TNP) derivatives obtained by means of trinitrobenzenesulfonic acid (TNBS). Unlike the procedures reported previously, we stopped the proteolysis reaction by boiling, and performed the trinitrophenylation in a 20% V. aqueous solution of MeCN.

Amines↗

Lidocaine kinetics: relationships between early lidocaine kinetics and indocyanine green clearance.

Lidocaine plasma levels and indocyanine green clearance were measured in five normal volunteers and eight patients admitted to the coronary care unit. All individuals received lidocaine as a 1 mg/kg bolus and a 35 microgram/kg/min constant infusion for 180 minutes. Eight of the 13 (62 per cent) individuals studied (all normal volunteers and three patients) developed early, potentially subtherapeutic plasma lidocaine levels (less than or equal to 2.4 microgram/ml) within 15 minutes after starting therapy. Those individuals with subtherapeutic levels had either minimal (American Heart Association Class) or no clinical evidence of congestive heart failure. The use of indocyanine green (ICG) clearance as an estimate of hepatic plasma flow showed that individuals with early, subtherapeutic lidocaine levels had higher ICG clearance (9.33 +/- 0.32 ml/min . kg versus 2.90 +/- 1.74 ml/min . kg) and shorter ICG t 1/2 (2.02 +/- 0.99 minutes versus 3.6 +/- 0.69 minutes) and larger volume of distribution (36.1 +/- 16.3 ml/kg versus 19.5 +/- 12.8 ml/kg) than patients without subtherapeutic levels. This study suggests that early lidocaine kinetics may be significantly altered by clinical conditions that alter hepatic blood flow. The reappearance of arrhythmias shortly after initiating lidocaine therapy in patients without heart failure may be due to rapid hepatic clearance and subtherapeutic blood levels rather than lidocaine-resistant arrhythmias.

Adult↗

Kinetic mechanism and pH dependence of the kinetic parameters of Pseudomonas aeruginosa phosphomannomutase/phosphoglucomutase.

The enzyme phosphomannomutase/phosphoglucomutase (PMM/PGM) is responsible for the formation of mannose 1-phosphate and glucose 1-phosphate in the human pathogenic bacterium Pseudomonas aeruginosa. Mannose 1-phosphate and glucose 1-phosphate are required for the biosynthesis of polysaccharides that contribute to the virulence of P. aeruginosa, so inhibitors of PMM/PGM may lead to clinically useful compounds. The V/K values for mannose 6-phosphate and glucose 6-phosphate show that they are equally good substrates for the enzyme. PMM/PGM overexpressed in Escherichia coli is isolated as a phosphoenzyme; surprisingly, mutation of serine 108 where phosphorylation occurs results in phosphorylation of a different residue so that activity is reduced only 20-fold from that of wild-type enzyme. In the reverse reaction glucose 1-phosphate exhibits substrate inhibition, which arises through its competition with the activator glucose 1,6-bisphosphate for binding to dephosphoenzyme. This phenomenon is consistent with a mechanism in which the enzyme phosphorylates the substrate to generate a bisphosphorylated intermediate that reorients in the active site to return its original phosphoryl group to the enzyme and generate the observed product. The pH dependence of the kinetic parameters suggests that the active site contains a residue that serves as a general base in the catalytic reaction and one that acts as a general acid. However, the pK of the general acid is 7.4 and that of the general base is 8.4 so these residues exist in a state of reverse protonation in the active enzyme.

Alginates↗

Cyclic kinetics and mathematical expression of the primary immune response. Cyclic kinetics of antibody-forming cells following immunization with Salmonella typhi O-antigen.

The kinetics of antibody-forming cells (AFC) in the spleen of rats immunized with Salmonella typhi O-antigen was investigated. The number of nucleated cells of spleen and blood serum antibody titres in passive haemagglutination were determined in parallel. Cyclic changes in the number of antibody-forming cells were detected as three peaks on the 4th, 9th, and 13th days following immunization. The fluctuations of their number were not related to the total number of nucleated cells of spleen. The antibody titres reached their peak on the 10th day following immunization, decreased by the 14th day and rose again on the 16th day after immunization. Repeated increases of the number of AFC were probably due to the regular, not accidental, recruitment of committed precursors cells (memory cells).

Animals↗

Cytochrome f and plastocyanin kinetics in Chlorella pyrenoidosa. I. Oxidation kinetics after a flash.

P-700, plastocyanin and cytochrome f redox kinetics were measured after one flash, using dark-adapted Chlorella in the presence of hydroxylamine and 3(3,4-dichlorophenyl)-1,1-dimethylurea. Plastocyanin becomes increasingly oxidized with a half-time of 70 microseconds, then undergoes reduction with a half-time of 7ms. Cytochrome f oxidation has a sigmoidal time-course and a half-time of 100 microseconds. Its redution exhibits a half-time of 4 ms. These results are interpreted in a linear scheme: (formula: see text). An equilibrium constant of 2 between cytochrome f and plastocyanin (PC), which contrasts with the large equilibrium constant between PC and P-700 is computed. The presence of cytochrome b6 in a cyclic path around Photosystem I is confirmed under these conditions.

Chlorella↗

The steady-state kinetic mechanism of ATP hydrolysis catalyzed by membrane-bound (Na+ + K+)-ATPase from ox brain. II. Kinetic characterization of phosphointermediates.

(1) The kinetics of the phosphorylated enzymic intermediates of (Na+ + K+)-ATPase from ox brain, which are formed by incubation of the enzyme with 25 microM AT32P, 150 mM Na+ and 1 mM Mg2+, have been studied in dephosphorylation experiments at 1 degree C. The dephosphorylation of the 32P-labelled enzyme was initiated by addition of either 1 mM unlabelled ATP, 2.5 mM ADP or 1 mM unlabelled ATP + ADP in concentrations from 25 to 1000 microM. (2) In the absence of ADP the dephosphorylation curve was linear in a semilogarithmic plot almost from t = 0, whereas by addition of ADP a biphasic behaviour was obtained. The slope of the slow phase of dephosphorylation was virtually independent of the ADP concentration. (3) The results were analysed by the mathematical equation corresponding to the simplest possible model for the interconversion and breakdown of the phosphointermediates: (formula: see text) where alpha, beta, H and G are functions of all the rate constants and H and G furthermore are functions of the initial values for [E1P] and [E2P]. (4) The analysis confirmed the model and enabled the determination of all the rate constants. (5) k-1 was found to be equal to k'-1 + k"-1 . [ADP] indicating an ADP-independent 'spontaneous' dephosphorylation of E1P. The rate constant for this process was close to that for dephosphorylation of E2P, i.e., k'-1 congruent to k3. Also the value of k"-1 was determined. (6) k3 was found to be at least 10 . k-2. The implication of this for the role of the E1P to E2P transition in the Na+ + K+)-stimulated ATP hydrolysis will be discussed in detail in the following paper (Plesner, I.W., Plesner, L., Nørby, J.G. and Klodos, I. (1981) Biochim. Biophys. Acta 643, 483--494). (7) A refinement of the model, accounting for the effect of Na+ on the steady-state ratio between [E1P] and [E2P] is proposed: (formula: see text). At [Na+] = 150 mM as used here, E1P(Na) and E'1P are assumed to be in rapid equilibrium. (8) Comparison of our results with those of others underlines the general validity of the conclusions of the present paper.

Adenosine Diphosphate↗

Kinetic analysis of duck epsilon-crystallin with L-lactate dehydrogenase activity: determination of kinetic constants and comparison of substrate specificity.

A systematic analysis of the kinetic properties of duck lens epsilon-crystallin with lactate dehydrogenase [LDH, (E.C. 1.1.1.27)] activity was carried out by employing some 19 different alpha-keto acids as substrates for this NADH-dependent LDH-catalyzed reaction. The steady-state Michaelis and catalytic constants (Km, kcat) were determined for a broad range of organic compounds. The results provide important insights regarding the binding and affinity of substrates to active sites of this enzyme crystallin and indicate a great potential for the application of the stable epsilon-crystallin as a catalyst to the synthesis of some important chiral alpha-hydroxyacids in a convenient and efficient way. It is also demonstrated for the first time that in addition to the enzymatic activity of lactate dehydrogenase, duck epsilon-crystallin also possesses the enzymatic activity of malate dehydrogenase.

Animals↗

Multicompartment kinetic models for lead. II. Linear kinetics and variable absorption in humans without excessive lead exposures.

Multicompartment models with constant fractional transfer rates have been fitted to experimental data on lead metabolism in four subjects studied by M. B. Rabinowitz, G. W. Wetherill, and J. D. Kopple (Science 182, 725-727, 1973; Environ. Health Perspect. 7, 145-153, 1974; Arch. Environ. Health 31, 220-223, 1976; J. Clin. Invest. 58, 260-270, 1976; J. Lab. Clin. Med. 90, 238-248, 1977). Long-term retention is estimated for blood, soft tissue, cortical and trabecular bone pools, and for facial hair. The absorption of lead from diet is shown to change with time, but no evidence was found for other variable or nonlinear kinetic mechanisms of lead metabolism in humans without excessive lead exposure.

Diffusion↗

Use of steady state kinetic methods to elucidate the kinetic and chemical mechanisms of retroviral proteases.

Despite the current plethora of structural data of HIV-1 protease and the availability of potent inhibitors, whose structures are based in part on the presumed mechanism of action of this enzyme, our actual understanding of its chemical mechanism has been until now based largely on the precedents of the mammalian and fungal aspartic proteases and static three-dimensional data. The available steady state kinetic data of the protease, as reviewed here, constitute a first step in a detailed description of the mechanism of the enzyme to complement the structural data.

Amino Acid Sequence↗

Kinetic model for membrane transport. 1. Effects of membrane volume and partitioning kinetics.

Equations for the transport of solutes through a membrane are derived, taking into account both the membrane volume and the partitioning kinetics, and have been found to involve two rate constants for solute transport, namely, those corresponding to solute transport from the solution to the membrane (k1) and from the membrane to the solution (k2). The time course followed before partitioning equilibrium has been attained, which is usually ignored, is shown to depend strongly on the relative magnitudes of k1 and k2.

Biological Transport↗

Kinetic study of the effects of calcium ions on cationic artichoke (Cynara scolymus L.) peroxidase: calcium binding, steady-state kinetics and reactions with hydrogen peroxide.

The apparent catalytic constant (k(cat)) of artichoke (Cynara scolymus L.) peroxidase (AKPC) with 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) increased 130-fold in the presence of calcium ions (Ca2+) but the affinity (K(m)) of the enzyme for ABTS was 500 times lower than for Ca2+-free AKPC. AKPC is known to exhibit an equilibrium between 6-aquo hexa-coordinate and penta-coordinate forms of the haem iron that is modulated by Ca2+ and affects compound I formation. Measurements of the Ca2+ dissociation constant (K(D)) were complicated by the water-association/dissociation equilibrium yielding a global value more than 1000 times too high. The value for the Ca2+ binding step alone has now been determined to be K(D) approximately 10 nM. AKPC-Ca2+ was more resistant to inactivation by hydrogen peroxide (H(2)O(2)) and exhibited increased catalase activity. An analysis of the complex H(2)O(2) concentration dependent kinetics of Ca2+-free AKPC is presented.

Calcium↗