Tyrosine protein kinase assays

Protein kinases form a large family of enzymes that play a major role in a number of live processes. The study of their action is important for the understanding of the transformation mechanisms and of the normal and pathological growth events. The quality of an enzyme assay is often the key point of an enzymatic study. It must be flexible and compatible with various experimental conditions, such as those for the purification process, the screening of inhibitors and the substrate specificity studies. As will be shown in the present review, two categories of substrates, peptidic and proteic, should be distinguished. The use of peptide substrates facilitates the determination of the recognition requirements of the enzyme and of the kinetic effects of even minute variations in their sequence. These linear peptide structures are assumed to mimic a complex interaction between the enzyme and a proteic substrate in which distant amino acids in the sequence are vicinal in the folded substrate. Less amenable to a systematic study, but probably more adequate to investigate the natural substrate of a given kinase, are the proteic substrates. Obviously the tools to measure protein kinase activities are not the same in these two cases. The main difficulty in assaying protein kinases is the use of labelles γ-ATP, mostly at large excess concentration, since the final product of the reaction has to be separated from the non-reacted labelled ATP. In the case of peptide substrates, the difficulty is to separate them from ATP basing on differences of molecular mass. Despite the efforts of many investigators to rely upon differences in solubility, in charges or in “affinity”, this separation, which is crucial for the assay, is still an unsolved experimental problem. Chromatographic, as well as electrophoretic assays appeared relatively late in this domain, and more work in assessing new methodologies might bring new breakthroughs in the next few years. Specific, simple and reliable kinase assays are still a major challenge. Their improvement will help to conduct specificity studies, to elucidate complex growth mechanisms in which they are involved and to discover more selective potent inihibitors.     

Ventilation and metabolism among rat strains

Strohl, Kingman P., Agnes J. Thomas, Pamela St. Jean, EvelynH. Schlenker, Richard J. Koletsky, and Nicholas J. Schork. Ventilation and metabolism among rat strains. J. Appl. Physiol. 82(1): 317-323, 1997.We examinedventilation and metabolism in four rat strains with variation in traitsfor body weight and/or blood pressure regulation.Sprague-Dawley [SD; 8 males (M), 8 females (F)], BrownNorway (BN; 10 M, 11 F), and Zucker (Z; 11 M, 12 F) rats were comparedwith Koletsky (K; 11 M, 11 F) rats. With the use of noninvasiveplethysmography, frequency, tidal volume, minute ventilation(E),O₂ consumption, andCO₂ production were derived atrest during normoxia (room air) and during the 5th minute of exposureto each of the following: hyperoxia (100% O₂), hypoxia (10%O₂-balanceN₂), and hypercapnia (7%CO₂-balance O₂). Statistical methods probedfor strain and sex effects, with covariant analysis by body weight,length, and body mass. During resting breathing, strain effects werefound with respect to both frequency (BN, Z > K, SD) and tidal volume(SD > BN, Z) but not to E. Sexinfluenced frequency (F > M) alone. Z rats had higher values forO₂ consumption,CO₂ production, and respiratoryquotient than the other three strains, with no independent effect bysex. During hyperoxia, frequency was greater in BN and Z than in SD orK rats; SD rats had a larger tidal volume than BN or Z rats; Z rats hada greater E than K rats; and M had alarger tidal volume than F. Strain differences persisted duringhypercapnia, with Z rats exhibiting the highest frequency andE values. During hypoxic exposure,strain effects were found to influenceE (SD > K, Z), frequency (BN > K), and tidal volume (SD > BN, K, Z). Body mass was only amodest predictor of E during normoxia, of both E and tidal volume withhypoxia, hypercapnia, or hyperoxia, and of frequency duringhypercapnia. We conclude that strain of rats, more than their body massor sex, has major and different influences on metabolism, the patternand level of ventilation during air breathing, and ventilation duringacute exposure to hypercapnia or hypoxia.

     

Apoptosis and nutrition: Involvement of amino acid transport system in repression of hybridoma cell death

Mouse hybridoma cells cultured on the verge of starvation-induced apoptosis, i.e. in a medium diluted with saline, proved to serve as a sensitive screening system for apoptosis-suppressing activity of nutrient medium components. Conventional amino acid mixtures were found to suppress the starvation-induced apoptosis, whereas a vitamin mixture was ineffective. (Frank F (1995) Biotechnol. Bioeng. 45: 86–90). Recent experiments showed that suppression of apoptosis, and concurrent resumption of growth, could be achieved by addition of single substances at millimolar concentrations. The set of active substances included certain coded L-amino acids (glycine, alanine, serine, threonine, proline, asparagine, glutamine, histidine), non-coded amino acids (-alanine, taurine, 4-aminobutyric acid), and a non-metabolizable analogue (2-aminoisobutyric acid). This finding shows that some amino acids do not act solely as nutrients, but also as specific signal molecules. The specificity of the effect points to the involvement of adaptively regulated amino acid transport systems A and N in maintaining the balance between triggering and suppression of starvation-induced apoptosis.     

Transport and control of Ca by pigeon erythrocytes. II. Evidence against a simple feedback control of cell Ca and evidence for the involvement of more than one pool

Cells were subjected to a range of ⁴⁵Ca²⁺ influx loads with A23187. We measured cell ⁴⁵Ca²⁺ with time and A23187 dose, and the apparent ⁴⁵Ca²⁺ influxes (≡“J(in,app)”) at Ca²⁺ steady state. We also measured endogeneous exchangeable and total cell Ca²⁺, which were 50 and 17–220 μM respectively. The effects of A23187 and Ca²⁺ on cell ATP, swelling, net Cl⁻ permeability, and cell morphology were measured. These were modest and do not affect our conclusions.J(in,app) 3 × 10⁻⁴ [A23187]^2.9·[Ca²⁺(o)]μmoles/l·min with 92–552 μM [Ca²⁺(o)] (≡ external Ca²⁺ concentration) and 0–7 μM A23187. J(in,app) was increased an order of magnitude by vanadate and is probably much less than the true influx. The least unlikely explanation found for the high [A23187] exponent, 2.9, was that most of the Ca²⁺ crossing the membrane is expelled by the pump before it can move deeper into the cell.Calcium pumping increased rapidly in response to increased influx, but the steady state cell ⁴⁵Ca²⁺ was approximately proportional to J(in,app) rather than approximately constant between 10 and 120 μmoles/l·min with 184 μM [Ca²⁺(o)]. This is not the result expected from a simple feedback mechanism.At high A23187 doses the pump appears fully activated resulting in a linear relation between cell/medium ⁴⁵Ca²⁺ and [A23187]⁻². From the plot we calculated α≡free/total exchangeable Ca²⁺ = 0.38 ± 0.08 (n = 3) and a maximum pump rate, “Pmax” = 78 μmole/l·min. Pmax is underestimated insofar as J(in,app) is less than the true influx.     

Effects of progestins and antiprogestins on mitochondria in uterine glandular cells in the rat

Summary The administration of progesterone to ovariectomized rats induces an increase in the volume density (Vv) of the mitochondria and the appearance of giant mitochondria in the uterine glandular cells. This experimental model, including a stereological analysis, allowed us to investigate and quantify a direct effect of progesterone on a well-defined cellular structure without the intervention of estrogen in a priming phase. Synthetic compounds, promegestone, gestrinone and RU 38486, were tested in this model either in place of progesterone or simultaneously with progesterone. The potent progestomimetic activity of promegestone was confirmed by the proliferation of giant mitochondria and a high Vv value for the mitochondria, the two other compounds being inactive even at higher doses. At lower doses, gestrinone and RU 38486 partially inhibit the action of progesterone and at higher doses they both show a complete antagonist effect by preventing the development of the mitochondria.     

Differentiation of cells producing polypeptide hormones (ACTH,MSH, LPH, α- and β-endorphin,GH and PRL) in the fetal porcine anterior pituitary

Summary The aim of the present study on the fetal porcine pituitary was (1) to detect by means of the immunoperoxidase technique the earliest stages of cells producing polypeptide hormones: -MSH, ACTH, -LPH, - and -endorphin, growth hormone (GH) and prolactin (PRL), (2) to study the development of the synthesis and the storage of these hormones during fetal life, and (3) to detect whether several hormones can be located in one and the same cell.The corticotropic cells were revealed as the earliest functional elements of the fetal anterior pituitary. Our results indicate clearly that ACTH, -MSH, -LPH, - and -endorphin appear at 34 days in the same regular, round or ovoid cells; no differences in the time of their appearance could be observed. The ACTH-cells, irregular or angular in shape and endowed with cytoplasmic processes such as described in the adult pituitary, were not seen until day 50. The first GH-cells were detected between 40 to 45 days of fetal life. From day 45 to 90, the GH-cells greatly increased in number and in staining intensity of their progressively extending cytoplasmic area, but they displayed the same regular and round shape. The PRL-cells were the last cell type to appear in the fetal pituitary. The first PRL-cells, small in size and round or ovoid in shape with a high nucleus/cytoplasm ratio, were detected at day 70. At day 80, the PRL-cells increased in size and staining intensity. They displayed an irregular elongated or stellated shape and cytoplasmic processes resembling those characteristic of the adult pituitary. These data suggest that in the fetal porcine pituitary: (1) ACTH, -LPH and related peptides are synthesized and stored in the same cells, and (2) PRL and GH appear in individual cellular elements.     

Discrete subaortic stenosis as part of a short stature syndrome

Summary Observations in a family point to the existence of autosomal dominant inheritance for discrete subaortic stenosis (DSS), which made up part of a multisystem disorder. Both parents, offspring of two full siblings, had short stature, obstructive lung disease (OLD), hoarseness and upturned nose. The father alone had aortic stenosis and inguinal hernia.The six offspring, aged from 13 to 28 years, were followed up for up to 8 years. While one of them was virtually normal, and one had only minor abnormalities, four siblings displayed clinical signs of progressive aortic stenosis. Of the two eldest siblings who eventually died, necropsy in one showed a discrete subaortic stenosis, which was hemodynamically proven in one and surgically corrected in another sibling.Upturned nose was present in each examined member of the family, short stature and hoarseness in five of the siblings, DSS in four, OLD, inguinal hernia and congested episcleral veins in three, kyphoscoliosis in two, while epicanthus, strabismus, microphthalmos and widely spaced teeth were noted in the deceased female. The prevalence of some of these traits in roughly three-quarters of the sibship was consistent with an underlying single gene abnormality in affected heterozygous parents. We proposed that this constitutes a new syndrome.     

A method for demonstrating the heterogeneity of pigeon red cell membrane vesicles based on their glycine transport activity

A procedure is described which is capable of detecting the changes in size and/or density of small membrane vesicles resulting from solute uptake. Vesicles which have taken up solute sediment more slowly in a density gradient, the ratio of glycine uptake/vesicle-trapped space is not uniform in the vesicle population, and vesicles with higher uptake/space ratios are preferentially retarded upon centrifugation.Alanine transport activity is associated with glycine transport activity in that retardation of vesicles due to glycine uptake equally retards vesicles possessing alanine uptake activity.     

Microcalorimetric evaluation of the efficacy of the alkaline treatment on the fermentation of straw

Summary A microcalorimetric study is proposed to follow the degradation of straw by a mixed bacterial culture. The effect of an alkali pretreatment of straw is described.