Molecular cloning of the cDNA for a growth factor-inducible gene with strong homology to S-100, a calcium-binding protein

We have identified a cDNA whose sequence is preferentially expressed when quiescent fibroblasts are stimulated to proliferate. The steady-state levels of the mRNA corresponding to this clone, called 2A9, are increased by serum, platelet-derived growth factor, and epidermal growth factor, but not by insulin or platelet-poor plasma. mRNA levels of 2A9 are also increased in human acute myeloid leukemia. The 2A9 cDNA has been molecularly cloned from an Okayama-Berg library, and its complete nucleotide sequence has been determined. It has an open reading frame of 270 nucleotides, which has a 55% homology with the coding sequence of the beta-subunit of the S-100 protein, a calcium-binding protein that belongs (like calmodulin and the vitamin D-dependent intestinal calcium-binding protein) to the family of calcium-modulated proteins and is found in abundance in several human tumors, including melanoma. The S-100 protein and the deduced aminoacid sequence of 2A9 are also partially homologous to the small subunit of a protein complex that serves as a cellular substrate to tyrosine kinase. The partial homology of 2A9 (whose RNA is inducible by growth factors and is overexpressed in human acute myeloid leukemias) to the S-100 protein, other calcium-modulated proteins, and the subunit of a substrate for tyrosine kinase, is particularly interesting in view of the role attributed to calcium and tyrosine kinases in the regulation of cell proliferation.     

Equilibrium binding of insulin to rat white fat cells at 15 degrees C

Equilibrium binding of insulin to isolated rat epididymal fat cells was investigated. A temperature of 15 degrees C was chosen for the study to minimize lysosomal degradation of insulin. Indeed, medium insulin lost only 1% of its precipitability in trichloroacetic acid during the 4-h incubation required to approach equilibrium. Binding was measured by a method that did not perturb the equilibrium of the system. A new formalism for analyzing binding data in general was introduced. A correction for trapping of insulin in the interstitial space of cell pellets was both necessary and sufficient to derive specific binding data from raw observations. Thus, so-called "nonspecific binding" was unmasked as a misnomer, and the expression "correction for trapping" was proposed as a substitute. Equations for one and two independent classes of binding sites were fit to the data by the method of maximum likelihood, and the best fit was selected based on Akaike's information criterion, as adapted for a constant fractional error. More than 99.7% of the binding sites were found to be describable by a simple binding isotherm with Kd,app = 8.8 multiplied by over divided by 1.3 nM. Less than 0.3% sites had a higher affinity (Kd approximately equal to 8 multiplied by over divided by 3 pM). There were 99,000 x/divided by 1.6 binding sites/cell. These equilibrium parameters are in agreement with values derived from a kinetic analysis, presented in the subsequent paper (Lipkin, E. W., Teller, D. C., and de Ha?n, C. (1986) J. Biol. Chem. 260, 1702-1711).     

Purification and characterization of two plasma membrane domains from ejaculated bull spermatozoa

Plasma membranes were detached from ejaculated bull spermatozoa by a brief sonication in a moderately hypotonic medium, and the released plasma membranes were partially purified by differential centrifugation. The resulting fraction was enriched 8- and 15-fold in alkaline phosphatase and 5' nucleotidase activities, respectively, compared with the starting sonicated spermatozoa. This total plasma membrane fraction was separated into two distinct fractions by equilibrium density centrifugation on a continuous linear sucrose gradient. Two peaks of light scattering material were formed at densities of 1.117 and 1.148 g/ml. The denser peak contained most of the protein of the plasma membrane fraction, whereas nearly all the concanavalin A binding activity was found in the lighter peak. The two bands had distinctly different polypeptide compositions when analyzed by SDS PAGE. Polyclonal antibodies were raised in rabbits against a major integral membrane glycoprotein of each fraction (Mr of 92,000 in the light peak and 98,000 in the dense peak). The two antigens were detected on the surface of intact spermatozoa by indirect immunofluorescence microscopy. The 92-kD protein (present in the lighter band) was detected only on the plasma membrane of the acrosomal and anterior postacrosomal regions of the head. The 98-kD antigen, present in the heavier band, was localized to the surface of the postacrosomal region of the head, to the principal piece of the tail, and to the connecting piece between the head and tail. The exclusive localization of the 92-kD polypeptide to the surface of the anterior portion of the head was confirmed by immunoelectron microscopy. These data show that the two fractions isolated on the sucrose gradient originate from different regions of the sperm cell plasma membrane.     

Rhizosphere acidification as a response to iron deficiency in bean plants

Iron deficiency in higher plants causes accumulation of salts of organic acids in the roots, the most characteristic being citrate. We show that citrate and malate accumulate in beans (Phaseolus vulgaris L. var Prélude), not because of a lack of the iron-containing enzyme aconitase (EC 4.2.1.3), but in close coupling to the extrusion of protons during rhizosphere acidification, one of the `Fe-efficiency' reactions of dicotyledonous plants. When proton excretion is induced in roots of control bean plants by addition of fusicoccin, only malate, not citrate, is accumulated. We propose that iron deficiency induces production of organic acids in the roots, which in beans leads to both proton excretion and an increased capacity to reduce ferric chelates via the induced electron transfer system in the root epidermis cells.     

Air pollution as a possible cause for the decline of some phanerogamic species in The Netherlands

In The Netherlands the decline of some phanerogamic species cannot be readily explained from obvious factors such as lowering of the groundwater table, eutrophication or land reclamation. For a number of species the hypothesis was tested that the decline is partly due to air pollution. A two-factor model was made in which decline is accounted for by (a) habitat destruction assessed from topographic maps and (b) air pollution measured as the SO₂ 95-percentile over the winter period 1978/1979. Effects of both factors were assumed to follow a sigmoid dose-effect curve. For a number of species decline proved to be significantly correlated with air pollution. These are notably species from the syntaxon Violion caninae. A comparison was made with results obtained for epiphytic lichens. It appears that for some phanerogamic species sensitivity is about the same as for moderately sensitive lichens.Nomenclature follows Heukels & van der Meijden (1983).Thanks are due to the Rijksherbarium, for providing some of their unpublished data; and to Ada Groeneveld, for technical assistance.     

Solubility of proteins

A theory is presented on the solubility of proteins, in the hydrated as well as in the dry state, and in water as well as in organic solvents. To this effect, colloidal stability is assimilated with the solubility of the proteins, considered as hydrated entities. By means of a surface thermodynamic approach it can be shown that an increase in size of a hydrated protein must lead to insolubility, even in the absence of any change in a protein's surface properties. This can be substantiated experimentally by comparing the surface properties of immune complexes with those of their constituent immunoglobulins, as well as by comparing some of the properties of intact tobacco mosaic virus with those of its monomeric capsid subunits. Insolubilization of proteins by means of charge interactions as well as by dehydration is studied; an explanation is given of why precipitation caused by charge interactions is more likely to lead to partial irreversible denaturation than precipitation caused by protein-protein interactions brought about by partial dehydration (e.g., by “salting-out”). A link is established between the smallness (or even the negative value) of the interfacial tension between given proteins and various solvents and their solubility in these solvents. The energy of hydration of proteins can also be measured, and the differences between the free energies of interaction of dried and hydrated proteins with water point toward the additional processes underlying the solubilization, i.e., toward the conformational change of a protein in the process of becoming hydrated. The parameter of conformational change of a protein, while becoming hydrated, appears to be more closely linked to its degree of hydration than to its hydration energy.     

Vitamin E deficiency and vitamin C supplements: exercise and mitochondrial oxidation

The effects of dietary antioxidant vitamins E and C on exercise endurance capacity and mitochondrial oxidation were investigated in rats. The endurance capacity of both vitamin E-deficient and vitamin C-supplemented, E-deficient rats was significantly (P less than 0.05) lower (38.1 and 33.6%, respectively) than control animals. Compared with the normal and vitamin E-deficient rats, there was a significant (P less than 0.05) increase in the concentration of vitamin C in blood and liver of the vitamin E-deficient, C-supplemented animals. Hence dietary vitamin C supplementation does not prevent the inhibition of exercise endurance capacity or increased hemolysis seen in vitamin E deficiency. The mitochondrial activities for the oxidation of palmitoyl carnitine and alpha-ketoglutarate were significantly (P less than 0.05) decreased by a single bout of exercise in brown adipose tissue but not in muscle, heart, or liver from vitamin C-supplemented, E-deficient groups of rats when compared with the activities in the tissue from the same group of rats killed at rest. Similar results were also seen in brown adipose tissue from vitamin E-deficient rats. The results suggest a tissue-specific role for vitamins E and C in substrate oxidation and show that the poor endurance capacity of vitamin E-deficient rats cannot be attributed to any changes in the mitochondrial activity in skeletal or cardiac muscles. It is also concluded that vitamin C supplementation, at least at the dose employed in the present study, cannot counteract the detrimental effects associated with vitamin E deficiency.     

Influence of ventrolateral surface of medulla on reflex tracheal constriction

To assess the role of structures located superficially near the ventrolateral surface of the medulla on the reflex constriction of tracheal smooth muscle that occurs when airway and pulmonary receptors are stimulated mechanically or chemically, experiments were conducted in alpha-chloralose-anesthetized, paralyzed, and artificially ventilated cats. Pressure changes within a bypassed segment of the trachea were used as an index of alterations smooth muscle tone. The effects of focal cooling of the intermediate areas or topically applied lidocaine on the ventral surface of the medulla on the response of the trachea to mechanical and chemical stimulation of airway receptors were examined. Atropine abolished tracheal constriction induced by mechanical stimulation of the carina or aerosolized histamine, showing that the responses were mediated over vagal pathways. Moderate cooling of the intermediate area (20 degrees C) or local application of lidocaine significantly decreased the tracheal constrictive response to mechanical activation of airway receptors. Furthermore, when the trachea was constricted by histamine, cooling of the intermediate area significantly diminished the increased tracheal tone, whereas rewarming restored tracheal tone to the previous level. These findings suggest that under the conditions of the experiments the ventral surface of the medulla plays an important role in constriction of the trachea by inputs from intrapulmonary receptors and in the modulation of parasympathetic outflow to airway smooth muscle.     

Structure and properties of enzyme graft copolymers: Temperature effects on HRP immobilization mechanism

The possibility of obtaining immobilized horseradish peroxidase (HRP) materials with K'(m) values close to that of the native enzyme, but with good thermal stability, was investigated. The photochemical reaction was used as the immobilization methodology. Temperature and catalyst concentration were found to be the main parameters able to control the immobilization reaction mechanism more than type of functional monomer, polymer-matrix, and enzyme-polymer ratios. By carrying out the immobilization reaction at 35 degrees C and using either bisacryloylpiperazine (BAP) or hexhydro-1,3,5-triacryloyl-s-triazine (HTsT) as the functional monomer, materials with a good thermal stabilization (the retained activity after 240 min at 60 degrees C was between 65-25%) as well as kinetic constants (0.6-0.8 x 10(-4)M) similar to that of the free enzyme (0.57 x 10(-4)M) were obtained. Since low K'(m) values were obtained also using a high polymer content (pBAP copolymers, 25%; pHTsT copolymers, 30%) and neither limitation to substrate diffusion nor a reduction of the enzyme mobility was found, the enzyme should be linked to the matrix during the last steps of monomer polymerization, and it should have an external disposition with respect to the support.     

Modifications evoked by piretanide in the mechanical activity of cardiovascular tissues

The effects of piretanide upon mechanical activity and pharmacological reactivity of vascular and myocardial tissues from normotensive rats were investigated. Magnitude of phasic contractions of isolated rat portal vein was diminished by the drug in a dose-related manner; contractile depression induced by piretanide (10(-4)M) was less in the presence of insulin (0.1 U/mL), glucose (22 mM) or pyruvate (5 mM). Responses to KCl (90 mM), or norepinephrine (2.5 X 10(-5)M) were also reduced. Contractile activity of atria and ventricle strips was diminished only when piretanide reached 10(-4)M. Results support direct actions of piretanide upon cardiac and vascular tissues. Possible mechanisms of action are discussed.