Isolation and Characterization of 2-Keto-3-Deoxyoctonate-Lipid A from a Heptose-Deficient Mutant of Escherichia coli

A heptose-deficient mutant of Escherichia coli has been isolated and from it a glycolipid, consisting of lipid A and 2-keto-3-deoxyoctonate (KDO), has been extracted with diisobutylketone-acetic acid-water. Based on beta-hydroxymyristic acid, the extractable glycolipid accounts for a major portion of the total lipid A in this mutant. A glycolipid, purified from the lipid extract by a combination of silicic acid and Sephadex LH-60 chromatography, contains glucosamine, phosphate, KDO, acetyl groups, and fatty acids in the following molar ratios: 1:2:2:1.7:5. These components account for over 80% of the lipid by weight. The fatty acid pattern of the glycolipid is typical of lipid A, the major component being beta-hydroxymyristic acid. The lipid also contains an amino sugar which appears to be 4-amino-4-deoxyarabinose. With the use of an ion-exchange paper chromatographic technique, gram-negative bacteria can be rapidly screened for the presence of this glycolipid. The mutant is believed to have a leaky defect in either biosynthesis of heptose or its incorporation into lipopolysaccharide. The lipopolysaccharide from the mutant contains only about a third as much heptose, glucose, and galactose as the parent CR34, a K-12 derivative. Chemical analysis and phage typing suggest that CR34 contains an incomplete core polysaccharide devoid of glucosamine.     

Self-regulation: A context for biofeedback

Editorial

Self-regulation: A context for biofeedback     

EFFECTS OF DIVISION-SYNCHRONIZING HYPOXIC AND HYPERTHERMIC SHOCKS UPON TETRAHYMENA RESPIRATION AND INTRACELLULAR ATP CONCENTRATION

The division of Tetrahymena pyriformis GL cells was synchronized with either seven hypoxic or five hyperthermic (heat) shocks. Hyperthermic shocks of 34°C produced no reduction in respiration rate and only a 19% decline in intracellular ATP concentration. Hypoxic shocks of 0.15% ambient oxygen concentration depressed intracellular ATP concentration 50%. It therefore appears that hypoxic shock, but not hyperthermic shock, reverses progress of Tetrahymena toward fission by reducing ATP concentration through a reduction of the rate of oxidative phosphorylation. After the first synchronized division, whether synchronized by intermittent hypoxia or hyperthermia, total respiration rate increased exponentially at the same rate of increase as total respiration rate in an exponentially growing (log phase) Tetrahymena cell culture. Before the first synchronized division, the total respiration rate increased exponentially but more slowly than after completion of the first synchronized division. The pattern of increase of total respiration during division synchronized by either procedure was different than the pattern of increase of total respiration of synchronous cells observed by Zeuthen.     

PERIODIC THERMAL STRESS: OPTIMAL SPACING PREDICTED BY A SIMPLE SET-BACK MODEL OF CELL DIVISION SYNCHRONIZATION

Digital computer simulations have been used to make quantitative predictions based on a simple set-back model of cell division synchronization. According to the model appropriate thermal stress reverses progress within a segment of the division cycle called the set-back interval. In the simulations normally distributed cell-to-cell variations in division cycling rate between periodic thermal shocks were produced with the Monte Carlo method.
The simulations have shown that reasonably good synchronization with the single shock per division strategy requires a relatively long set-back interval and small cell-to-cell variations in rate of progress through the division cycle. The simulations have shown that the degree of synchrony produced by such periodic shocks is highly dependent on the time interval between shocks—with a series of as many as seven shocks inappropriately spaced producing less synchrony than a single shock! The optimal time interval between successive thermal shocks was found approximately equal to the mode division cycle time at synchrony equilibrium multiplied by 1 plus half the fraction of the division cycle occupied by the set-back interval. Position of the set-back interval within the division cycle had little effect on synchrony at the end of the final shock.     

Beta-adrenergic receptor-mediated phospholipase C activation independent of cAMP formation in turkey erythrocyte membranes.

The effect of the beta-adrenergic receptor agonist isoproterenol on guanine nucleotide-dependent phospholipase C (PLC) activity was examined in turkey erythrocyte membranes prepared from [3H]inositol-labeled turkey erythrocytes. In the presence of guanosine 5'-(gamma-thiotriphosphate) (GTP[S]) isoproterenol caused a dose-dependent stimulation of [3H]inositol phosphate ([3H]InsP) formation. The activation of PLC by GTP[S] occurred after an initial lag period of 1-2 min and was followed by a sustained rate of [3H]InsP formation which remained linear for 4-5 min. Isoproterenol decreased the lag period for GTP[S]-induced [3H]InsP formation and increased PLC activity at all time points following this lag. Consequently, isoproterenol shifted the dose-response curve for GTP[S] to the left (10-fold) and increased the maximal response. The EC50 value for isoproterenol-induced activation of PLC was 104 +/- 17 nM. Isoproterenol also potentiated GTP-dependent PLC activity but was ineffective in stimulating the enzyme in the presence of AIF4-. The PLC activation by isoproterenol was completely inhibited by propanolol and atenolol but was unaffected by prazosin or yohimbine. Although GTP[S] and isoproterenol could increase cAMP formation in this membrane preparation, the isoproterenol-induced stimulation of PLC occurred in the absence of ATP and was independent of cAMP formation. Furthermore, addition of cAMP, 8-bromo-cAMP, forskolin, or either the regulatory or catalytic subunits of cAMP-dependent protein kinase failed to stimulate [3H]InsP formation and had no effect on the responses elicited by GTP[S] and isoproterenol. Isoproterenol also stimulated [3H]InsP2 and [3H]InsP3 production in intact erythrocytes. Cholera toxin had no effect on [3H]InsP formation in the intact cells under conditions where it stimulated cAMP accumulation. In addition, the activation of PLC by GTP[S] and isoproterenol was unaffected in membranes prepared from cholera toxin-treated erythrocytes. These data demonstrate that stimulation of turkey erythrocyte beta-adrenergic receptors by isoproterenol results in a direct activation of guanine nucleotide-dependent PLC.     

Effect of 1-oleoyl-2-acetylglycerol and other lipids on phosphatidylcholine synthesis and cholinephosphate cytidylyltransferase activity in cultured type II pneumocytes

We previously reported that addition of phosphatidylglycerol to the culture medium stimulates phosphatidylcholine synthesis and cholinephosphate cytidylyltransferase activity in type II pneumocytes. In view of the known biological effects of diacylglycerols and since phosphatidylglycerol could be metabolized to diacylglycerol, we now examined the effects of diacylglycerols on the same parameters. The rate of choline incorporation into phosphatidylcholine was increased 30-60% by 10 microM phosphatidylglycerol, diolein, mixed diacylglycerols and 1-oleoyl-2-acetylglycerol (OAG). The effects of phosphatidylglycerol and OAG were not additive, suggesting a similar mechanism of action. The diacylglycerols and phosphatidylglycerol increased the activity of cholinephosphate cytidylyltransferase in type II cell sonicates by 35-50%, but had no effect on the activities of choline kinase, cholinephosphotransferase or 1-acylglycerophosphocholine acyltransferase. Again, the effects of OAG and phosphatidylglycerol on cytidylyltransferase were not additive. It is known that addition of lipids to the assay mixture increases the activity of cholinephosphate cytidylyltransferase in vitro and inclusion of the above lipids (1.1 mM) in the in vitro assay mixture increased cytidylyltransferase activity in type II cell sonicates. In addition, the stimulatory effects of OAG and of diolein, as well as of phosphatidylglycerol as reported previously, in the culture medium on cytidylyltransferase activity in type II cells were diminished or abolished when the assay was carried out in the presence of sufficient amounts of the same lipids to stimulate maximally the activity in vitro. These data show that lipids in the culture medium stimulate phosphatidylcholine biosynthesis in type II cells by direct activation of cholinephosphate cytidylyltransferase.     

Effects of glucocorticoid and thyroid hormones on regulatory enzymes of fatty acid synthesis and glycogen metabolism in developing fetal rat lung

Although glucocorticoid and thyroid hormones are known to act synergistically to stimulate surfactant production, they have opposite effects on other parameters of fetal lung maturation. We recently reported that the developmental increases in de novo fatty acid synthesis and glycogen accumulation in fetal rat lung were accelerated by dexamethasone but prevented by triiodothyronine and that the dexamethasone-induced increases were diminished when the two hormones were administered together. We have now examined the effects of maternal administration of these hormones on activities of enzymes of lung fatty acid synthesis and glycogen metabolism in the rat. There was a developmental increase in fatty-acid synthase activity between 19 and 21 days gestation. This activity was increased by dexamethasone but decreased by triiodothyronine. When the two hormones were administered together the stimulatory effect of dexamethasone was decreased from 56% to 29%. The stimulatory effect on fatty-acid synthase was also observed in fetal lung explants cultured in the presence of dexamethasone. This shows that the effect of the hormone was directly on the fetal lung. Dexamethasone had no effect on liver fatty-acid synthase. There was a developmental decrease in acetyl-CoA carboxylase activity but it was not affected by the hormones. These data show that the developmental and hormone-induced changes in fetal lung de novo fatty acid synthesis are mediated by fatty-acid synthase. Although there were developmental changes in fetal lung 6-phosphofructokinase, glycogen synthase and glycogen phosphorylase activities, these enzymes were not affected by the hormones.     

Cutting edge: a novel viral TNF receptor superfamily member in virulent strains of human cytomegalovirus.

The UL144 open reading frame found in clinical isolates of human CMV (HCMV) encodes a structural homologue of the herpesvirus entry mediator, a member of the TNFR superfamily. UL144 is a type I transmembrane glycoprotein that is expressed early after infection of fibroblasts; however, it is retained intracellularly. A YXXZ motif in the highly conserved cytoplasmic tail contributes to UL144 subcellular distribution. The finding that no known ligand of the TNF family binds UL144 suggests that its mechanism of action is distinct from other known viral immune evasion genes. Specific Abs to UL144 can be detected in the serum of a subset of HCMV seropositive individuals infected with HIV. This work establishes a novel molecular link between the TNF superfamily and herpesvirus that may contribute to the ability of HCMV to escape immune clearance.     

Fitting fluorescence emission spectra of probes bound to biological membranes

We show that fluorescence emission spectra for molecules containing the dansyl fluorophor can be accurately described as skewed Gaussians, and that spectra for dansyl probes bound to biological membranes can be resolved using least-squares techniques into two components, representing probe bound to the lipid and protein sites in the membrane.     

A benefit-finding intervention for family caregivers of persons with Alzheimer disease: study protocol of a randomized controlled trial

Background

Patients with ST-elevation myocardial infarction (STEMI) not treated with primary or rescue percutaneous coronary intervention (PCI) are at risk for recurrent ischemia, especially when viability in the infarct-area is present. Therefore, an invasive strategy with PCI of the infarct-related coronary artery in patients with viability would reduce the occurrence of a composite end point of death, reinfarction, or unstable angina (UA).

Methods

Patients admitted with an (sub)acute myocardial infarction, who were not treated by primary or rescue PCI, and who were stable during the first 48 hours after the acute event, were screened for the study. Eventually, we randomly assigned 216 patients with viability (demonstrated with low-dose dobutamine echocardiography) to an invasive or a conservative strategy. In the invasive strategy stenting of the infarct-related coronary artery was intended with abciximab as adjunct treatment. Seventy-five (75) patients without viability served as registry group. The primary endpoint was the composite of death from any cause, recurrent myocardial infarction (MI) and unstable angina at one year. As secondary endpoint the need for (repeat) revascularization procedures and anginal status were recorded.

Results

The primary combined endpoint of death, recurrent MI and unstable angina was 7.5% (8/106) in the invasive group and 17.3% (19/110) in the conservative group (Hazard ratio 0.42; 95% confidence interval [CI] 0.18-0.96; p = 0.032). During follow up revascularization-procedures were performed in 6.6% (7/106) in the invasive group and 31.8% (35/110) in the conservative group (Hazard ratio 0.18; 95% CI 0.13-0.43; p < 0.0001). A low rate of recurrent ischemia was found in the non-viable group (5.4%) in comparison to the viable-conservative group (14.5%). (Hazard-ratio 0.35; 95% CI 0.17-1.00; p = 0.051).

Conclusion

We demonstrated that after acute MI (treated with thrombolysis or without reperfusion therapy) patients with viability in the infarct-area benefit from a strategy of early in-hospital stenting of the infarct-related coronary artery. This treatment results in a long-term uneventful clinical course. The study confirmed the low risk of recurrent ischemia in patients without viability.

Trial registration

ClinicalTrials.gov: NCT00149591.