Inhibition of stimulated osmotic water flow by fluphenazine, a calmodulin inhibitor, in the isolated toad skin

The effects of fluphenazine, a phenothiazine calmodulin inhibitor, on the osmotic water flow (Posm) across isolated skins of Bufo arenarum toads were tested. Fluphenazine inhibited the increase in Posm induced by agents known to act through the cyclic AMP system (oxytocin, db-cAMP, theophylline, KCl and isoproterenol). The inhibitory effect was faster and more intense when the drug was present in the epidermal bath, and persisted after rinsing the preparation for 100 min. Our results indicate that phenothiazine's action may be primarily exerted at a site distal to cyclic AMP generation.     

Reducing errors in the accident department: a simple method using radiographers.

The assessments by radiographers of 1628 consecutive patients referred for radiography in the casualty department were analysed. The radiographers missed abnormalities in the radiographs in 68 of the cases. Casualty officers missed abnormalities in 63 cases, but only 35 patients were common to both groups. Twenty eight of the radiographs interpreted wrongly by casualty officers were interpreted correctly by radiographers; 16 of these 28 were thought by the accident and emergency consultant to be clinically important. It is suggested that a system whereby radiographers signal abnormalities should be standard practice.     

Crystallization and preliminary X-ray study of a complex between d(ATGCAT) and actinomycin D

Crystals of a 2:1 complex between the self-complementary DNA hexamer d(ATGCAT) and the antitumor drug actinomycin D have been grown from solutions of polyethylene glycol 400. The crystals are orthorhombic with space group P2(1)2(1)2(1) and a = 95.6, b = 42.7, and c = 40.8 A. A Patterson map calculated from preliminary diffractometer data as well as packing considerations suggest a model in which the actinomycin D is intercalated into a double-stranded DNA hexamer. There are four such complexes in the asymmetric unit.     

Structural and conformational analysis of sialyloligosaccharides using carbon-13 nuclear magnetic resonance spectroscopy

The analysis of the carbon-13 chemical shift data of NeuAc alpha (2----3)Gal beta (1----4)Glc and NeuAc alpha (2----3)Gla beta-(1----4)GlcNAc and their respective NeuAc alpha (2----6) isomers established distinct and different conformations of the sialic acid residue, depending on the type of anomeric linkage [alpha-(2----3) vs. alpha (2----6)]. Interactions between the NeuAc residue and the Glc or GlcNAc residue are particularly strong in the case of the alpha (2----6) isomers. Similar effects are observed for the larger oligosaccharides [II3(NeuAc)2Lac and IV6NeuAcLcOse4] and even in intact glycoproteins and polysaccharides. It is proposed that the NeuAc alpha (2----3) isomers assume an extended conformation with the sialic residue at the end (terminal) of the oligosaccharide chain or branch. The NeuAc alpha (2----6) isomers are assumed to be folded back toward the inner core sugar residues.     

A comparison of chromium-51 and iron-59 for estimating erythrocyte survival in the cat

Erythrocyte survival studies were conducted on eight, normal, healthy, 1-year-old male specific-pathogen-free cats using both chromium-51 and iron-59 simultaneously. The chromium-51 procedure gave a half-life value of 11.1 +/- 0.9 days. This was considerably lower than would be expected on the basis of the experimentally determined iron-59 erythrocyte survival time of 51.2 +/- 14.9 days. The results of this study indicated that there was considerable loss of the chromium-51 label in the cat other than that from senescence alone. An analysis of the chromium-51 disappearance curve indicated that there were two exponential disappearance rates for the chromium-51 label and, in the absence of cell death, approximately 67% of the label was lost with a rate constant of 0.02 per day and 33% was lost with a rate constant of 0.1 per day. An equation is presented which models the loss of chromium-51 label which could be used to calculate erythrocyte survival from a chromium-51 disappearance curve. Blood volume measurements, hemograms, bone marrow differential results, and iron kinetic values also were determined and the results presented. While a reasonable approximation of the erythrocyte life span could be made by correcting the chromium-51 values for losses other than senescence, the iron-59 procedure would be the preferred method in cats.     

Transient kinetic analysis of turnover-dependent fluorescence of 2',3'-O-(2,4,6-trinitrophenyl)-ATP bound to Ca2+-ATPase of sarcoplasmic reticulum

The fluorescence of 2',3'-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP) bound to the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum is greatly enhanced during turnover induced by ATP plus Ca2+ (Watanabe, T., and Inesi, G. (1982) J. Biol. Chem. 257, 11510-11516). We have studied the kinetics of induction of TNP-ATP fluorescence and of its decay and have found a close correlation with levels of phosphorylated intermediate of the enzyme, E-P. Steady-state kinetic studies suggested competitive binding of ATP and TNP-ATP to the catalytic site, with Km and Ki values of 2.4 and 1.0 microM, respectively. Rate constants for fluorescence enhancement and for E-P formation in the presteady state were 1.2 s-1 or 97-130 s-1 under conditions resulting in TNP-ATP or ATP saturation respectively, of the enzyme at inception of reaction. The slow process was concluded to be the koff for dissociation of TNP-ATP from the catalytic site. Following this dissociation, a second TNP-ATP site was detected, which both formed (97-130 s-1) and decayed (0.22 s-1) synchronously with E-P. TNP-ATP binding to this noncatalytic site was rapid (5 X 10(7) M-1 s-1) and resulted in high fluorescence during steady-state turnover. Fluorescence was found to be dissociated from E-P by KCl (100 mM). KCl had little effect on E-P levels, but decreased fluorescence by 68%. These studies provide independent kinetic evidence for the existence of both catalytic and noncatalytic, or "regulatory," nucleotide-binding sites, but cannot distinguish whether the two sites exist independently or whether the catalytic site is transformed into a regulatory site on phosphorylation. The latter site, which shows relatively high selectivity for TNP-ATP over ATP, and which is simultaneously hydrophobic and freely accessible to the medium, may play a role during energy transduction. The changes occurring at this site during catalysis are conveniently monitored with TNP-ATP fluorescence.     

Nitrate reductase: an improved assay method for phytoplankton

A new assay for measuring the activity of nitrate reductasein phytoplankton, based upon the permeability of cells treatedwith toluene to substrates and products, is described. The methodis simple and, since the reaction is carried out directly ona glass fiber filter, can be easily performed in the field oron shipboard. In comparison with previous methods, this techniquegave higher absolute amounts of NO^–₂ formed per unit tuneand higher enzymatic activities per sample volume when testedwith axenic algal cultures and with natural phytoplankton populationsfrom Lake Kinneret, the River Jordan and the Eastern Mediterranean.     

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).     

Redescription of Scapholeberis echinulata Sars 1903 (Crustacea,Cladocera)

Scapholeberis echinulata Sars is rediscribed, based on type material of Sars (S. echinulata), Daday (s. erinaceus) and the author's own samples. A differential diagnosis with Scapholeberis spinifera Nicolet and S. mucronata O. F. Müller, S. microcephala Sars, S. kingi Sars and S. aurita Fischer is given.