A fluorescence method for estimation of toxemia: binding capacity of lipoproteins and albumin in plasma.

The hazard of toxemia, a condition resulting from the spread of toxins by the bloodstream, is regulated by plasma proteins capable of binding with free toxins. As toxin binding results in a reduction of available binding sites, measuring the proteins' binding capacity can be used to estimate toxemia severity. Suggested by this approach, a novel fluorescence method was developed to determine lipoprotein and albumin binding capacities in whole plasma. The method entails two steps: specific binding of N(n-carboxy)phenylimide-4-dimethyl-aminonaphthalic acid with albumin followed by addition of 12-(9-anthroyloxy)stearic acid which, under these conditions, binds mostly with lipoprotein. Reduced fluorescence intensity of the probes in plasma of patients compared to that of healthy donors reflected saturation of binding sites by toxins, thereby estimating toxemia severity. Poor correlation was found between the lipoprotein and albumin binding abilities, suggesting their independent diagnostic values. The simplicity and rapidity of this method are advantageous for its clinical application.

Involvement of the oxygen storage protein myoglobin in muscle damage under oxidative stress.

Myoglobin (Mb), the muscular oxygen reservoir, was shown to possess peroxidative reactivity in presence of H2O2 leading to oxidation of isolated cellular proteins like myosin. The objective of this study was to investigate the peroxidative effect of Mb/H2O2 on proteins in intact myofibrils (MF). Incubation of chicken leg MF in isotonic, pH 7.3 buffer at 37 degrees C in the presence of Mb (30 microM) and H2O2 (200 microM), resulted in aggregation of MF material as inspected under light microscope. SDS-PAGE analysis revealed presence of high molecular weight aggregates at the expense of myosin heavy chains, but not actin. This crosslinking was unaffected by S-S reducing agents. Continuous low flow (0.03-3.00 microM/minute), produced by glucose oxidase and glucose, was more active than bolus H2O2 addition in myosin crosslinking in MF material. Hemin which may be released from Mb under oxidative stress, was more active than Mb as a trigger of MF peroxidative aggregation. Calcium-ATPase activity of crosslinked MF was considerably lost. These findings suggest that Mb/H2O2 may lead to oxidation of neighbouring muscular protein thereby jeopardize their functioning thus explaining muscular malfunction under oxidative stress.

Combined effects of ATP and its analogs on the membrane permeability in transformed mouse fibroblasts.

Extracellular ATP (0.6 mM) induces a marked decrease in the membrane potential, followed by an increase in cell membrane permeability in transformed mouse fibroblasts. The effects of the ATP analogs, p[CH2]ppA and p[NH]ppA (0.6 mM), on the membrane potential and permeability are much less pronounced. ATP at 0.05 mM has no effect by itself, but markedly increases the analog-induced membrane potential dissipation and permeability. The data suggest that ATP-induced membrane permeation is composed of two processes: One is common to ATP and its analogs and appears to be a receptor-mediated process. The second is unique for ATP, effective even at low concentration (0.05 mM), and might be mediated by cell surface enzymes, for which ATP, but not its analogs, serves as a substrate.

Surgical closed pulmonary valvotomy for critical pulmonary stenosis: implications for the balloon valvuloplasty era.

BACKGROUND: Closed pulmonary valvotomy for critical pulmonary stenosis has no apparent advantage over the percutaneous balloon technique, though it is used when balloon valvuloplasty fails. Experience of this technique at the Heart Institute, Tel Hashomer, since it was first used in 1973 has been reviewed. METHODS: Thirty eight infants up to 1 year old (25 of them neonates--that is, nil to 1 month old) with critical pulmonary stenosis were operated on from 1973 to 1989. All had a transventricular valvotomy, by a modification of the Brock method, and all underwent cardiac catheterisation before surgery. RESULTS: Five of the 25 neonates (20%) died, but none of the other infants, so that the total mortality (five out of 38) was 13%. Three of the 38 required an aortopulmonary shunt. All 38 survivors were followed up--from one month to 14 years (mean 7.5 years). All were symptom free at the last check up. Fifteen of the survivors had required further surgery; this was successful in all cases. CONCLUSIONS: For the balloon valvuloplasty era surgical pulmonary valvotomy provides a good back up for failed attempts at percutaneous valvuloplasty. Review of outcome provides data for comparison with balloon valvuloplasty in the future.

Purification and characterization of acetyl esterase from bull testes.

Acetyl esterase (acetic-ester acetylhydrolase, EC 3.1.1.6) from bull testes was purified 325-fold by ammonium sulphate precipitation, chromatography on DEAE-cellulose, hydroxyapatite and finally, gel filtration on a Sephadex G-200 column. The purified enzyme appeared as a single protein band on native polyacrylamide gel electrophoresis and in isoelectric focusing (pI 5.25). In both methods, the activity coincided with the protein band. A single protein band corresponding to Mr 70,000 was obtained by SDS-polyacrylamide gel electrophoresis. The reported amino-acid composition indicates that the enzyme contains three half-cystine residues, of which only one could be detected, by titration, as a free -SH group. No free amino terminal was detected by dansylation.

ATP analogues induce membrane permeabilization in transformed mouse fibroblasts.

The mechanism underlying ATP-induced permeabilization of transformed mouse fibroblasts was studied by using nonhydrolyzable analogues of ATP. Incubation of 3T6 cells with 0.6 mM of either ATP, 5'-adenylyl imidodiphosphate (p[NH]ppA) or adenosine 5'-[beta, gamma-methylene]triphosphate (p[CH2]ppA) resulted in an increase of 17-, 8- or 5-times, respectively, in the cell membrane permeability, measured by the efflux of normally impermeant metabolites from the cells. The induced cell permeabilization was preceded by a reduction in the membrane potential (delta psi), determined according to the distribution of the cation tetraphenylphosphonium (TPP+) between the cells and the medium. Reduction of 26, 18 and 13 mV in delta psi was exerted by 0.6 mM of either ATP, p[NH]ppA or p[CH2]ppA, respectively. In 3T3 cells the untransformed counterparts of 3T6 cells, neither reduction of delta psi, nor alterations in membrane permeability were exerted by either ATP or by its analogues. The data indicate that the dissociation of the beta, gamma-phosphate bond is not essential for membrane permeabilization by external ATP, implying that the binding of ATP to the cell surface of transformed cells is sufficient to initiate the permeabilization process. The data also suggest that delta psi is involved in the control of membrane permeability.

Interaction of nucleotides and cations with the (Ca2+, Mg2+)-ATPase of sarcoplasmic reticulum as determined by fluorescence changes of bound 1-anilino-8-naphthalenesulfonate.

The changes in fluorescence of 1-anilino-8-naphthalenesulfonate (ANS-) have been used to determine binding of ligands to the (Ca2+, Mg2+)-ATPase of sarcoplasmic reticulum vesicles, isolated from rabbit skeletal muscle. ANS- binds to sarcoplasmic reticulum membranes with an apparent Kd of 3.8 X 10(-5) M. The binding of ANS- had no effect on Ca2+ transport or Ca2+-dependent ATPase activity. EGTA, by binding endogenous Ca2+, increased the fluorescence intensity of bound ANS- by 10-12%. Subsequent addition of ATP, ADP, or Ca2+, in the presence or absence of Mg2+, reversed this change of fluorescence. The binding parameters, as determined by these decreases in fluorescence intensity, were as follows: for ATP, Kd = 1.0 X 10(-5) M, nH = 0.80; for ADP, Kd = 1.2 X 10(-5) M, nH = 0.89; and for Ca2+, Kd = 3.4 X 10(-7) M, nH = 1.8. The binding parameters for ITP and for the nonhydrolyzable analogue, adenyl-5'-yl-beta, gamma-methylene)diphosphate, were similar to those of ATP, but GDP, IDP, CDP, AMP, and cAMP had lower apparent affinities. Millimolar concentrations of pyrophosphate also decreased the fluorescence of bound ANS-, whereas orthophosphate caused a small (2-3%) increase in fluorescence in Ca2+-free media. Vanadate, in the presence of EGTA, decreased the fluorescence of bound ANS-with half-maximal effect at 4 X 10(-5) M. The changes of fluorescence intensity of bound ANS- appear to reflect conformational changes of the (Ca2+, Mg2+)-ATPase, consequent to ligand binding, with the low and high fluorescence intensity species corresponding to the E1 and E2 conformations, respectively. These appear to reflect similar conformational states of the (Ca2+, Mg2+)-ATPase to those reported by changes in intrinsic tryptophan fluorescence (DuPont, Y. (1976) Biochem, Biophys. Res. Commun. 71, 544-550).