A common mechanism for activation of the Na+/H+ exchanger by different types of stimuli

The mechanism of activation of Na+/H+ exchanger by various stimuli was studied in the human epidermoid carcinoma cell line A431 and in peripheral blood mononuclear cells (PBM). Intracellular pH (pHi) was measured by using the fluorescent dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. Stimulation of A431 cells by epidermal growth factor (EGF), bradykinin (BK), phorbol-12-myristate 13-acetate (PMA), and osmotic shrinkage resulted in exchanger activation. In PBM, activation of Na+/H+ exchanger was induced by concanavalin A (Con A) and phytohemagglutinin (PHA), as well as PMA and osmotic shrinkage. Inhibition of protein kinase C inhibited only PMA-stimulated exchanger activation in both cell types. When osmotic shrinkage was applied after exposure of the cells to any agonist, augmentation of exchanger activation by osmotic stress was observed. These findings suggest that various stimuli activate Na+/H+ exchanger through different mechanisms. Kinetic analysis demonstrated that activation of the exchanger by any type of stimulus resulted in modification of the apparent affinities for intracellular H+ (H+i) and intracellular Na+ (Na+i) in opposite directions. While there is an increased apparent affinity for H+i, the apparent affinity for Na+i decreases. This finding suggests that in A431 cells this phenomenon serves as a common mechanism for activation of Na+/H+ exchanger by different stimuli.     

A new red cell discriminant incorporating volume dispersion for differentiating iron deficiency anemia from thalassemia minor

A novel red cell discriminant function [MCV2 x RDW/(Hgb x 100)] was compared to six other discriminants in 102 patients with established mild iron deficiency anemia and 33 patients with beta-thalassemia minor. The discriminant incorporates the two key measurements of erythrocyte cell volume distribution, namely the mean (MCV) and standard deviation (RDW), which are known to be helpful for distinguishing between these two frequent causes of microcytic hypochromic anemia. Data used for the learning set to develop the new discriminant were obtained using an electrical impedance automated whole blood analyzer (Coulter S + IV) and were applied as a validation set for six other discriminants. The discriminant was also tested on smaller subsets of the patients groups using data obtained on either an alternate electrical impedance instrument (Sysmex E-5000) or a laser light scattering based system (Technicon H*1). From the comparison it was concluded that use of a discriminant function that incorporates a measurement of red cell volume dispersion results in enhanced accuracy for distinguishing iron deficiency anemia from thalassemia minor.     

Guanylyl cyclase is a heat-stable enterotoxin receptor.

Plasma membrane forms of guanylyl cyclase have been shown to function as natriuretic peptide receptors. We describe a new clone (GC-C) encoding a guanylyl cyclase receptor for heat-stable enterotoxin. GC-C encodes a protein containing an extracellular amino acid sequence divergent from that of previously cloned guanylyl cyclases; however, the protein retains the intracellular protein kinase-like and cyclase catalytic domains. Expression of GC-C in COS-7 cells results in high guanylyl cyclase activity. In addition, heat-stable enterotoxin from E. coli, but not natriuretic peptides, causes marked elevations of cyclic GMP and is specifically bound by cells transfected with GC-C. The enterotoxin fails to elevate cyclic GMP in nontransfected cells or in cells transfected with the natriuretic peptide/guanylyl cyclase receptors. These results show that a heat-stable enterotoxin receptor responsible for acute diarrhea is a plasma membrane form of guanylyl cyclase.     

Effect of vascular cannulation on serum concentrations of LH, FSH, prolactin and cortisol in prepubertal gilts

Two experiments were conducted to determine whether cannulation of the jugular vein in gilts alters serum concentrations of LH, FSH, prolactin (PRL) or cortisol (C). In Experiment 1, 12 crossbred prepubertal gilts weighing 95 +/- 1.3 kg were immobilized by snaring, and tygon tubing was threaded into the anterior vena cava through a 12-gauge needle inserted into the jugular vein. Five hours later, blood samples were drawn at 20-min intervals for 4 h (Day 0). Samples were also drawn at 20-min intervals for 4-h periods 24 h (Day 1) and 48 h (Day 2) after cannulation. Serum concentrations of LH were similar (P=0.26) among Day 0 (0.40 ng/ml), Day 1 (0.39 ng/ml) and Day 2 (0.34 ng/ml). Serum PRL was similar (P=0.07) among Day 0 (4.10 ng/ml), Day 1 (3.87 ng/ml) and Day 2 (3.43 ng/ml). Serum concentrations of C were greater (P < 0.001) on Day 0 (8.32 ng/ml) than Day 1 (4.48 ng/ml) or Day 2 (3.54 ng/ml). In Experiment 2, cannulas were placed in 29 prepubertal gilts. Two days after initial cannulation, six blood samples were drawn at 20-min intervals. Gilts were then immobilized by snaring, and a second cannulae was inserted into the contralateral vein. Five blood samples were taken at 2-min intervals during the second cannulation and then six samples were drawn at 20-min intervals. Serum LH and FSH were not altered by cannulation or elevated during the subsequent 2-h sampling period (P>0.05). In contrast, serum concentrations of PRL rose slowly (P<0.05) during cannulation and remained elevated for 60 min before returning to baseline. Serum concentrations of C rose within 6 min of cannulation, remained elevated for 30 min, and then declined over the next 90 min. From these two experiments, it appears that secretory patterns of LH and FSH can be accurately assessed immediately after cannulation in prepubertal gilts. Measurements of serum PRL and C that reflect nonstressed conditions, however, cannot be obtained until at least 2 h or 1 d after cannulation, respectively.     

Mutational analysis of autonomously functioning trans-activating peptides encoded by adenovirus E1A: evidence for two subdomains.

We have shown previously that a chemically synthesized adenovirus E1A region 3 peptide of 49 amino acids, protein domain 3 (PD3; residues 140 to 188 of the 289-amino-acid protein), trans activates viral genes in vitro and in vivo. To study structure-function relationships, we synthesized N-terminal deletion and cysteine substitution mutant peptides and tested their activities in a cell microinjection assay. Peptides lacking 1 to 12 N-terminal residues exhibited 5- to 50-fold-reduced molar specific activities, whereas those lacking 16 or 18 residues were inactive. Substitution of each of five PD3 cysteine residues with alanine resulted in substantial losses of activity: mutants in the PD3 N-terminal portion showed 40 to 55% of wild-type activity but required a 20-fold-higher concentration than PD3, whereas those in the C-terminal half were as much less active. These peptide mutant studies suggest the existence of two PD3 functional regions: one, localized in the C-terminal 70 to 75% of the molecule, is essential for trans activation; the other, localized in the N-terminal 25 to 30%, can be overridden to a significant extent at high peptide concentrations.     

Specificity, stability, and potency of monocyclic beta-lactam inhibitors of human leucocyte elastase.

Stable, potent, highly specific, time-dependent monocyclic beta-lactam inhibitors of human leucocyte elastase (HLE) are described. The heavily substituted beta-lactams are stable under physiological conditions including in the presence of enzymes of the digestive tract. The beta-lactams were unstable in base. At pH 11.3 and 37 degrees C they were hydrolyzed with half-lives of 1.5-2 h. Hydrolysis produced characteristic products including the substituent originally at C-4 of the lactam ring, a substituted urea, and products resulting from decarboxylation of the acid after ring opening. The most potent beta-lactam displayed only 2-fold less activity versus HLE than alpha 1PI, the natural proteinaceous inhibitor. The compounds were more potent against the human and primate PMN elastases than versus either the dog or rat enzymes. Differences in the structure-activity relationships of the human versus the rat enzymes suggest significant differences between these two functionally similar enzymes. The specificity of these compounds toward HLE versus porcine pancreatic elastase (PPE) is consistent with the differences in substrate specificity reported for these enzymes [Zimmerman & Ashe (1977) Biochim. Biophys. Acta 480, 241-245]. These differences suggest that the alkyl substitutions at C-3 of the lactam ring bind in the S1 specificity pocket of these enzymes. The dependence of the stereochemistry at C-4 suggests additional differences between HLE and PPE. Most of the compounds do not inhibit other esterases or human proteases. Weak, time-dependent inhibition of human cathepsin G and alpha-chymotrypsin by one compound suggested a binding mode to these enzymes that places the N-1 substitution in the S1 pocket.