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A J Waring

Publications and source records attributed to A J Waring.

At least 73 records · Page 4Linked to original sources
PubMed1990

Structure-function relationships of bovine pulmonary surfactant proteins: SP-B and SP-C.

Pulmonary surfactant contains at least three unique proteins: SP-A, SP-B and SP-C. SP-B and SP-C from bovine surfactant are markedly hydrophobic and have molecular masses between 3 and 26 kDa. We identify surfactant proteins under nonreducing conditions on polyacrylamide gels with approximate molecular mass of 5, 14, 26 kDa (SP-5, 14, 26) when organic solvent-soluble material is eluted from a Sephadex LH-20 size exclusion column followed by separation on a high-performance reverse-phase chromatography system. These bands correspond to monomeric SP-C, oligomeric SP-C and oligomeric SP-B, respectively. Computer analysis (Eisenberg-hydrophobic moment) of sequences for these proteins suggests that SP-B contains surface-seeking amphiphilic segments. In contrast, SP-C resembles a more hydrophobic transmembrane anchoring peptide. Dispersions containing dipalmitoylphosphatidylcholine, phosphatidylglycerol, palmitic acid and multimeric SP-B and SP-C duplicate the surface activity of natural surfactant when assayed in a pulsating bubble surfactometer. We speculate that oligomers of SP-B and monomers and oligomers of SP-C may act cooperatively in affecting surfactant function. An important function of SP-B and SP-C may be to affect the ordering of surfactant lipids so that rates of transport of surfactant lipids to the hypophase surface in the alveoli are enhanced.

Amino Acid Sequence↗
PubMed1990

A prototype retractor system designed to minimize ischemic brain retractor injury: initial observations.

The authors have developed and patented a neurosurgical retractor system incorporating an infrared emitter and detector that allows detection of cerebral pulsations. Gentle contact with the surface of cat brains shows cerebral pulsations that correlate with arterial pulse as well as mechanical ventilation. The amplitude of cerebral pulsations decreases with higher retraction pressure and disappears at approximately 20 mmHg. The pressure on the surface of the brain decreased 50% in 5 minutes even though the position of the retractor was maintained constant. The authors postulate that monitoring cerebral pulsation may prove useful in clinical neurosurgery with respect to avoiding excessive retraction, which causes brain damage.

Animals↗
PubMed1989

Studies of the slowing of the dienone-phenol rearrangement of a steroidal 1,4-dien-3-one by the introduction of an 11-keto group.

It has been shown previously that steroidal 11-keto-1,4-dien-3-ones undergo dienone-phenol rearrangement significantly more slowly than their 11-deoxy-, or 11 beta-hydroxy- and 11-acetoxy analogs. We have tested the explanations suggested for this, and are able to reject most of them. There remains the probability that the 11-keto group reduces the migrating ability of the adjacent atom, C-9, which frequently migrates in steroidal rearrangements.

Androstenes↗
PubMed1987

Effect of ethanol on amylase secretion and cellular calcium homeostasis in pancreatic acini from normal and ethanol-fed rats.

The effects of ethanol on stimulus-secretion coupling were assessed by studying amylase release, Ca2+-homeostasis, and changes in physical properties of membranes in isolated rat pancreatic acini. In acini from normal rats, ethanol (50 mM and above) in vitro caused a dose-dependent stimulation of amylase release and an increase in cytosolic free Ca2+ concentration. Ethanol did not affect amylase secretion stimulated by cholecystokinin-octapeptide (CCK8), a secretagogue that acts by increasing cytosolic free Ca2+ levels, but did potentiate the secretion of amylase induced by vasoactive intestinal peptide (VIP) which raises intracellular cAMP. Ethanol also increased the rate of 45Ca2+ exchange. In acini labeled with the spin-probe 12-doxyl stearic acid, ethanol disordered the pancreatic plasma membranes. By contrast, in acini from animals that had chronically (6-7 weeks) ingested ethanol, the membranes were resistant to this disordering effect of ethanol. Chronic ethanol feeding lowered the total cellular calcium content and ionophore (A23187)-releasable pools of acinar calcium (11 and 24% respectively), and led to a 15-30% decrease in the rate of 45Ca2+ exchange. Chronic ethanol ingestion also lowered the basal rate of amylase secretion, but ethanol in vitro stimulated amylase secretion more than in control preparations. However, these differences in basal and ethanol-induced amylase secretion were not accompanied by corresponding changes in intracellular free Ca2+. The data suggest that ethanol perturbs cell membranes and also disturbs cellular Ca2+ homeostasis. These effects may explain its actions as a weak Ca2+-mediated secretagogue. However, the membrane alterations induced by chronic ethanol feeding do not prevent the ethanol-induced interference with cellular calcium homeostasis.

Amylases↗
PubMed1987

The molecular dynamics of actin measured by a spin probe attached to lysine.

Rabbit skeletal muscle G-actin was labeled with a spin probe, 3-(5-fluoro-2,4-dinitroanilino)proxyl. Tryptic digestion of the labeled actin followed by ultrafiltration and ion-exchange column chromatography indicated that the label was attached to residue Lys-61. This residue is found within a 9-kDa N-terminal segment that is easily degraded by proteolytic enzymes. The rate of reduction of the nitroxide bond by ascorbate was measured to determine the accessibility of the probe to small molecules in the solvent. These experiments showed that label bound to G-actin was relatively inaccessible to ascorbate, suggesting that it is buried within the protein structure. Polymerization further decreased the accessibility of the probe. Replacing bound Ca2+ with Mn2+ decreased the observed intensity of the electron paramagnetic resonance signal, indicating the spin label is about 2 nm distant from the metal binding site on the actin molecule. Labels attached to G-actin displayed an absorption spectrum characteristic of rotational motion with a correlation time (tau c) of 7 X 10(-9) s, which is faster than that for the whole molecule. Labels attached to F-actin had a value of tau c, measured using saturation transfer electron paramagnetic resonance, of 2 X 10(-5) s, which shows that the probe has a greater degree of mobility than the filament. The binding of heavy meromyosin or troponin-tropomyosin to labeled actin resulted in a further increase in the rotational correlation times, with the greatest decrease in mobility (tau c = 1 X 10(-4) s) observed when both were bound. Together the above results suggest that the 9-kDa segment of actin is mobile relative to the rest of the molecule and that this mobility can be influenced by the binding of heavymeromyosin or troponin-tropomyosin.

Actins↗
PubMed1986

Abnormal membrane properties of the sarcoplasmic reticulum of pigs susceptible to malignant hyperthermia: modes of action of halothane, caffeine, dantrolene, and two other drugs.

The role of sarcoplasmic reticulum (SR) in malignant hyperthermia (MH) was studied using the heavy microsomal fraction prepared from semitendinosus muscles of both normal and genetically MH-susceptible pigs. In the presence of ATP, SR was loaded with 70 nmol Ca2+/mg SR protein. Under these conditions, MH-SR demonstrated Ca2+-induced Ca2+ release (Ca-ICaR) and halothane-induced Ca2+ release (halothane-ICaR; halothane concentrations as low as 10 microM). Normal SR did not demonstrate these release phenomena. Dantrolene inhibited the halothane-ICaR, but did not inhibit the Ca-ICaR. Ruthenium red and tetracaine inhibited both types of Ca2+ release. From the measurement of passive Ca2+ efflux, it was shown that dantrolene did not affect the Ca2+ permeability of the SR itself, but suppressed only the halothane-induced increment of the permeability. The membrane order parameter of the SR, as measured by the spin-probe EPR technique, indicated that halothane disordered the lipid bilayer of MH-SR to a greater extent than it did of normal SR. This halothane disordering effect on MH-SR was antagonized by dantrolene. Ruthenium red and tetracaine did not antagonize the halothane disordering effect. These results raise the possibility that halothane could disturb the structure of the lipoprotein complex in MH-SR in such a way that it could open the Ca2+-release channels. The Ca2+ thus released further opens the channel through the Ca-ICaR mechanism in a positive feedback fashion, thus triggering the MH syndrome. The efficacy of dantrolene in ameliorating the MH syndrome might be related to the inhibition of this halothane effect.

Animals↗
PubMed1985

Characterization by electron paramagnetic resonance and studies on subunit location and assembly of the iron-sulfur clusters of Bacillus subtilis succinate dehydrogenase.

Succinate dehydrogenase is a conserved membrane-bound enzyme consisting of two nonidentical subunits: a flavo iron-sulfur protein (Fp) subunit, containing a covalently bound flavin, and an iron-sulfur protein (Ip) subunit. Bacillus subtilis succinate dehydrogenase in wild type bacteria and 12 well characterized succinate dehydrogenase-defective mutants were examined by low temperature EPR spectroscopy to characterize the enzyme and study subunit location and biosynthesis of its iron-sulfur clusters. The wild type B. subtilis enzyme contains iron-sulfur clusters which are analogous to clusters S-1 and S-3 of bovine heart succinate dehydrogenase but with slightly different EPR characteristics. Spins from cluster S-2 were not detectable as in the case of the intact form of bovine heart succinate dehydrogenase. However, dithionite reduction of the B. subtilis enzyme greatly enhanced spin relaxation of the ferredoxin-type cluster S-1, indicating the presence of the cluster S-2. Iron-sulfur cluster S-1 was found to be assembled in soluble succinate dehydrogenase subunits in the cytoplasm, but only if full-length Fp polypeptides and relatively large fragments of Ip polypeptides were present. Cluster S-1 was not detected in mutants with soluble mutated Fp polypeptides or in a mutant totally lacking Ip subunit polypeptide. Iron-sulfur clusters S-1, S-2, and S-3 were assembled also when the covalently bound flavin in the Fp subunit was absent. Clusters S-1 and S-3 in the membrane-bound flavin-deficient succinate dehydrogenase were not reduced by succinate but could be reduced by electron transfer from NADH dehydrogenase via the menaquinone pool.

Bacillus subtilis↗
PubMed1985

Sarcoplasmic reticulum membrane of rat skeletal muscle is disordered with chronic alcohol ingestion.

Heavy sarcoplasmic reticulum (SR) was prepared from skeletal muscle of chronic alcoholic rats and control rats. Compared with control rat SR, the SR prepared from alcoholic rats is leakier to Ca2+ and has a lower level of maximum storable Ca2+. With in vitro exposure to ethanol, the Ca2+ release of alcoholic rat SR was not enhanced as much as that of SR prepared from control rats. By using the spin-probes 5-, 7-, 12-, and 16-doxylstearic acid, the disordering effect of ethanol on the SR membrane was studied. The results suggest that SR membrane of chronic alcoholic rats is less ordered than that of control rats even in the absence of in vitro ethanol. However, chronic SR membrane shows a greater resistance to the disordering effect of in vitro addition of ethanol than control rat SR.

Alcoholism↗
PubMed1984

Liver ischemia increases the molecular order of microsomal membranes by increasing the cholesterol-to-phospholipid ratio.

An accelerated degradation of phospholipid is the likely basis of irreversible cell injury in ischemia, and the membranes of the endoplasmic reticulum of the liver are a convenient system with which to study the effect of such a disturbance on the structure and function of cellular membranes. In the present report, electron spin resonance spectroscopy has been used to evaluate changes in the molecular ordering of microsomal membrane phospholipids in the attempt to relate the loss of lipid to alterations in membrane structure. The order parameter, S, was calculated from spectra reflecting the anisotropic motion of 12-doxyl stearic acid incorporated into normal and 3-h ischemic microsomal membranes. Over the temperature range 4-40 degrees C, the molecular order (S) of ischemic membranes was increased by 8-10%. This increase was reproduced in the ordering of the phospholipids in liposomes prepared from total lipid extracts of the same membranes. In contrast, after removal of the neutral lipids, liposomes prepared from phospholipids of ischemic and control membranes had the same molecular order. There were no differences in the phospholipid species of control and ischemic membranes or in the fatty acid composition of the phospholipids. In the neutral lipid fraction of ischemic membranes, however, triglycerides and cholesterol were increased compared to control preparations. There were no free fatty acids. The total cholesterol content of the liver was unchanged after 3 h of ischemia. The cholesterol-to-phospholipid ratio of ischemic membranes, however, was increased by 22% from 0.258 to 0.315 as a consequence of the loss of phospholipid. Addition of cholesterol to the control total lipid extracts to give a cholesterol-to-phospholipid ratio the same as in ischemic membranes resulted in liposomes with order parameters similar to those of liposomes prepared from ischemic total lipids. It is concluded that the degradation of the phospholipids of the microsomal membrane results in a relative increase in the cholesterol-to-phospholipid ratio. This is accompanied, in turn, by an increased molecular order of the residual membrane phospholipids.

Animals↗
PubMed1984

Effects of chronic ethanol consumption on the partition of lipophilic compounds into erythrocyte membranes.

We have previously reported that chronic ethanol intoxication decreases the binding of various lipophilic substances to membranes of synaptosome and hepatic mitochondria. To determine whether this is a widespread effect, we used the quenching of the steady state fluorescence of membrane-bound diphenylhexatriene by halothane and the partitioning spin probe, 5-doxyl decane, to estimate the concentration of these compounds in erythrocyte plasma membranes. Membranes from ethanol-fed rats were resistant to the quenching of fluorescence by halothane or halothane plus 5-doxyl decane, compared with those from control animals, indicating lower quencher concentrations in the membranes of treated rats. These findings were confirmed by direct measurement of the partition of radiolabeled halothane into the membranes and by direct determination of the partition parameter of 5-doxyl decane by electron paramagnetic resonance spectroscopy. Halothane decreased the fluorescence lifetime to a larger extent in control membranes than in those from ethanol-fed rats. When various effects of halothane were expressed as a function of the concentration of halothane in the membrane, rather than the external buffer, no differences between membranes from control and treated rats were found. These data demonstrate that chronic ethanol intoxication decreases the partition of certain lipophilic compounds into the plasma membranes of erythrocytes, but that, at equivalent concentrations within the membrane, the interactions of halothane and the dye are comparable in both types of membranes. We suggest that the decreased partition of lipophilic compounds into biologic membranes is a widespread adaptive response to chronic ethanol intoxication and may play an important role in the resistance of such membranes to disordering after chronic ethanol ingestion.

Animals↗
PubMed1982

Chronic ethanol ingestion increases calcium uptake and resistance to molecular disordering by ethanol in liver microsomes.

Calcium uptake and molecular ordering were studied in hepatic microsomes from rats treated chronically with ethanol and were compared with the effects of ethanol in vitro. Calcium uptake was increased by 30% in microsomes from ethanol-fed animals, the Vmax being increased and the Km remaining unchanged. In Arrhenius plots of calcium uptake, the transition temperature in the 25-30 degrees C range was higher by 1.5 degrees C in microsomes from ethanol-fed rats. Chronic ethanol administration resulted in more rigid microsomal membranes, as evidenced by EPR spectra of the spin probe 5-doxylstearic acid. In a plot of the order parameter, S, as a function of temperature, microsomes from ethanol-fed rats showed a discontinuity at a higher temperature than those from controls (29.9 versus 24.2 degrees C). Ethanol, in vitro, inhibited microsomal calcium uptake in both preparations. However, the degree of inhibition was greater in controls. The rate of calcium uptake by microsomes from ethanol-treated animals in the presence of 100 mM ethanol was the same as those from controls in the absence of ethanol. In vitro, increasing concentrations of ethanol progressively decreased molecular ordering of control microsomes, whereas little change was noted in microsomes after chronic ethanol treatment.

Adenosine Triphosphate↗
PubMed1982

Rotational motion of cytochrome c derivatives bound to membranes measured by fluorescence and phosphorescence anisotropy.

Molecular motion of metal-free and metal-substituted cytochrome c derivatives was examined using the anisotropy of emissions from the singlet and the triplet states. The anisotropy of fluorescence provides a means to study the motion of cytochrome c in the nanosecond time scale, since the fluorescence lifetime of metal-free cytochrome c is around 10 ns. We find that the anisotropy of fluorescence of metal-free cytochrome c when bound to mitochondria does not decay, but when bound to phospholipids has a small component which decays independently of the rotation of the whole molecule. The use of phosphorescence extends the time scale for study into the millisecond regime, since the lifetime of the excited triplet state of zinc cytochrome c, as measured by triplet-triplet absorption and phosphorescence emission is approximately equal to 9 ms for free zinc cytochrome c and 7 ms for mitochondrial membrane-bound zinc cytochrome c at room temperature. The decay of anisotropy of phosphorescence emission of mitochondrial membrane-bound zinc cytochrome c is clearly biphasic; the fast component corresponds to a rotational relaxation time of 300 mus and the slow component with relaxation time of approximately equal to 6 ms. The slow component appears to be due to the rotation of the entire mitochondrion, whereas the fast component was interpreted to be due to the rotation of cytochrome c in a cone about a single axis perpendicular to the plane of the membrane surface.

Animals↗
PubMed1981

Synthesis and degradation of cyclic nucleotides in the pituitary gland, ovary and testis of rats treated with D-Trp6-luteinizing hormone-releasing hormone.

The effect of administration of D-Trp6-Luteinizing Hormone-Releasing Hormone (LH-RH) on synthesis and degradation of cyclic nucleotides was studied in the rat. There were no significant changes in the rate of synthesis and degradation of cyclic AMP in the ovary, testis and pituitary gland of D-Trp6-LH-RH-treated rats as compared to controls. On the other hand, the levels of cyclic GMP and activity of guanylate cyclase were significantly higher in the ovary and testis as well as in the pituitary gland of animals which received the analog. The rate of hydrolysis of cyclic GMP was unchanged by the administration of D-Trp6-LH-RH. Interestingly, the cyclic CMP phosphodiesterase seemed to be activated in animals treated with D-Trp6-LH-RH.

Animals↗
PubMed1981

Membranes and phospholipids of liver mitochondria from chronic alcoholic rats are resistant to membrane disordering by alcohol.

Using the spin probe 5-doxylstearic acid, we studied the structural perturbations of rat liver mitochondrial membranes produced by exposure to ethanol in vitro and by chronic ethanol feeding. The addition of ethanol in vitro to mitochondria from control animals appears to "fluidize" the membranes, as evidenced by a pronounced decrease in the order parameter. By contrast, in membranes from rats fed ethanol chronically, there was no effect on the order parameter. This resistance of the mitochondrial membranes from chronically intoxicated animals to the fluidizing effect of ethanol probably results from a change in the composition of the phospholipids, because the same differential response to ethanol was observed upon using vesicles of mitochondrial phospholipids extracted from control and chronically treated rats. In the presence of 0.025--0.1 M ethanol, a range that prevails in the blood of chronic alcoholics, the order parameter of mitochondrial membranes from rats fed ethanol was comparable to that of control membranes without ethanol in vitro. Analysis of extracted mitochondrial phospholipids showed that the cardiolipin from ethanol-fed animals had fatty acyl residues that are more saturated than those of controls. These findings point to the underlying molecular mechanism of our previous observation that mitochondria from chronic alcoholic rats are more resistant to uncoupling by ethanol at physiological temperature [Rottenberg, H., Robertson, D. E. & Rubin, E. (1980) Lab. Invest. 42, 318--326]. We suggest that an adaptive change in the phospholipid composition leads to structural alterations, which result in increased resistance to disruption of mitochondrial membranes by ethanol. These changes in lipid composition and structure may explain many, if not all, of the mitochondrial abnormalities that have been previously reported to result from chronic ethanol intoxication.

Alcoholism↗