NMR studies of the structure and stability of the 1 : 2 actinomycin D-d-pG-C complex in aqueous solution

Bacteriophage-resistant mutants isolated and classified in a previous study were examined for alterations in their lipopolysaccharide (LPS) composition, and properties likely to be affected by alterations in LPS composition were studied. It was found that many of the mutants of the Ktw (K2-resistance), Ttk (T2, T4, or K19 resistance), Bar (bacteriophage), Wrm (wide-range mutants), and miscellaneous resistance groups were altered in their response to a series of antibiotics and to two LPS-specific bacteriophages, C21 and U3. Furthermore, many of the bacteriophages to which these mutants were resistant adsorbed to LPS preparations. By direct sugar analysis of the mutant LPS preparations, it was shown that the mutants fitted into six distinct classes, which are readily derived from LPS core with a structure resembling that of Salmonella or Escherichia coli O100. A number of the mutants were shown to map between pyrE and mtl, which has been previously shown to be the site of a cluster of rfa genes in both Salmonella and E. coli. Outer membrane protein composition was studied in the above mutants using polyacrylamide gel electrophoresis. Some strains were shown to have alterations in the amount of major proteins. The nature of the bacteriophage receptors involved and the alterations leading to resistance are discussed.     

Two-dimensional 1H and 15N NMR titration studies of hisactophilin

We have used two-dimensional 1H-15N heteronuclear single quantum correlation spectroscopy to measure the pH dependence of backbone amide group chemical shifts in the actin binding protein hisactophilin over the pH range 5.7-11.1. Most of the resonances can be analyzed using a simple equation involving a single apparent ionization constant, pK(app). The majority of resonances in the protein titrate with pK(app) values of 5.6-7.4. The results can be rationalized in terms of titration of many histidine residues in hisactophilin. The titration data provide direct experimental support for the proposed models of the atomic basis of actin and membrane binding by hisactophilin.     

1H NMR spectroscopy studies of Huntington's disease: correlations with CAG repeat numbers

Huntington's disease (HD) is the result of an expanded (CAG) repeat in a gene on chromosome 4. A consequence of the gene defect may be progressive impairment of energy metabolism. We previously showed increased occipital cortex lactate in HD using localized 1H spectroscopy. We have now extended these studies to show an almost threefold elevation in occipital cortex lactate in 31 HD patients as compared with 17 normal control subjects (p < 10(-11)). The spectra in three presymptomatic gene-positive patients were identical to normal control subjects in cortical regions, but three in eight showed elevated lactate in the striatum. Similar to recently reported increases in task-related activation of the striatum in the dominant hemisphere, we found that striatal lactate levels in HD patients were markedly asymmetric (higher on the left side). Markers of neuronal degeneration, decreased N-acetylaspartate (NAA)/creatine and increased choline/creatine levels, were symmetric. Both decreased NAA and increased lactate in the striatum significantly correlated with duration of symptoms. When divided by his or her age, an individual's striatal NAA loss and lactate increase were found to directly correlate with the subject's CAG repeat number, with correlation coefficients of 0.8 and 0.7, respectively. Similar correlations were noted between postmortem cell loss and age versus CAG repeat length. Together, these data provide further evidence for an interaction between neuronal activation and a defect in energy metabolism in HD that may extend to presymptomatic subjects.     

Dexamethasone and dexamethasone phosphate detected by 1H and 19F NMR spectroscopy in the aqueous humour

To apply nuclear magnetic resonance (NMR) spectroscopy to study the penetration of dexamethasone phosphate into the aqueous humour from rabbit following topical administration. After topical administration of 0.1%, 1.0% and 10% dexamethasone phosphate solutions, respectively, samples of aqueous humour were aspirated, freeze-dried, redissolved in deuterium oxide and analyzed by high resolution 1H and 19F NMR spectroscopy. In order to study the lipophilic and hydrophilic metabolites of the drug, samples obtained after application of 1% dexamethasone phosphate were extracted with methanol/chloroform, and then extracted with perchloric acid. In all samples obtained from eyes denuded of the corneal epithelium prior to administration of dexamethasone, signals corresponding to the chemical shifts of the drug were identified in 19F NMR spectra. In the experiments performed with 1% dexamethasone phosphate, both dexamethasone and dexamethasone phosphate were detected in the aqueous humour. Using 10% dexamethasone phosphate solutions, signals from the drug were detected in 1H NMR spectra simultaneously with signals from about twenty other substances present in the aqueous humour. NMR spectroscopy appears to be a valuable method for studying dexamethasone metabolism and penetration into ocular tissues. It provides simultaneous detection of both the drug metabolites and other substances in the sample and might offer a complementary approach to other analytical methods.     

2H NMR studies of a myristoylated peptide in neutral and acidic phospholipid bicelles

Deuterium NMR spectra of Myr-d27-GNAAAAKKGSEQES (Cat14), the N-terminal 14-residue peptide from the catalytic subunit of cAMP-dependent protein kinase A (PKA), illustrate how magnetically aligned neutral and acidic phospholipid bicelles can be used to characterize the ordering and mode of binding of peptides to membranes. Since Cat14 is electrically neutral, the major interaction responsible for binding is the insertion of the myristoyl group into the hydrophobic core of the bilayer. The inclusion of 25% phosphatidylserine or phosphatidylglycerol into phosphatidylcholine bicelles results in a moderate increase in the ordering of the peptide relative to the bicelle normal, presumably because of favorable electrostatic interactions between the phospholipid headgroups and the two lysines in positions 7 and 8. Successful preparation of acidic bicelles was achieved by careful adjustment of lipid composition, pH and ionic strength.     

Measurement of molecular association in drug: cyclodextrin inclusion complexes with improved 1H NMR studies

The molecular association of haloperidol with hydroxypropyl-beta-cyclodextrin, expressed by the binding constant of the inclusion complex formed, was calculated from the changes on the 1H NMR spectra of the drug in the presence of the cyclodextrin. The stoichiometry of the complex was calculated by use of the continuous variation method (Job plot), and found to be 1:1. The binding constant for the 1:1 complex was calculated using improved non-linear models, which were solved by non-linear least-squares regression analysis, applying an iteration procedure. Three improved mathematical models for more accurate calculation of binding constants are proposed. The models are free from any assumptions and from practical or theoretical shortcomings.     

Signalling initiated with CD4-TCR or TCR-TCR interactions: comparison of tyrosine phosphorylation patterns and CD45 effects

LY 300164 [7-acetyl-5-(4-aminophenyl)-8,9-dihydro-8-methyl-7H-1,3-dioxolo(4, 5H)-2,3-benzodiazepine], administered intraperitoneally up to 2 mg/kg, did not influence the threshold for electroconvulsions. In doses of 2.5-4 mg/kg, LY 300164 significantly raised the threshold. In subprotective doses against electroconvulsions, this excitatory amino acid receptor antagonist enhanced the protective activity of intraperitoneally given valproate, carbamazepine and diphenylhydantoin against maximal electroshock-induced convulsions in mice. The anticonvulsive action of phenobarbital was potentiated by LY 300164 only at 2 mg/kg. The non-N-methyl-D-aspartate receptor antagonist did not affect the plasma levels of the antiepileptic drugs, so a pharmacokinetic interaction is not probable. Combined treatment with LY 300164 (2 mg/kg) and the antiepileptics studied (providing 50% protection against maximal electroshock) did not impair the motor performance of mice, evaluated in the chimney test. Valproate, at its ED50 of 280 mg/kg against maximal electroshock, produced motor impairment. As shown in the passive avoidance task, combination of LY 300164 (2 mg/kg) with valproate or diphenylhydantoin resulted in impairment of long-term memory. Alone among the antiepileptics, valproate (280 mg/kg) and phenobarbital (28.5 mg/kg) disturbed long-term memory. The results suggest that blockade of glutamate-mediated events via non-NMDA receptors leads to enhancement of the anticonvulsive activity of conventional antiepileptics. Some combinations of LY 300164 with antiepileptic drugs were superior to these antiepileptics alone in terms of their lack of adverse effects.     

Water and solute activities in the aqueous H2SO4 -(NH4)2SO4 solutions

The activities of water in H₂SO₄-(NH₄)₂SO₄-H₂O solutions, the concentrations of which ranged from 0.1 to 4.0 mol kg_-1 H₂SO₄ and 0.1 to 5.0 mol kg_-1 (NH₄)₂SO₄, were determined by an isopiestic method at 298 K. The experimentally determined water activities showed considerable deviation from the Zdanovskii rule. The activities of water were not in agreement with those calculated from the Robinson-Bower equation, even though the formation of sulfato-complexes of NH₄(I) and the dissociation of sulfuric acid were taken into consideration. The behavior of H₂SO₄-(NH4)₂SO₄ solu-tions is quite different from other systems. Water activities of aqueous solutions of H₂SO₄-Li₂SO₄ and H₂SO₄-Na₂SO₄ can be calculated from the Robinson-Bower equation with satisfactory accuracy. The interesting behavior of (NH₄)₂SO₄ can be attributed to the water structure-breaking property of NH₄ ⁺ ions. An additivity equation for estimating the water activity of aqueous H₂SO₄ solutions con-taining (NH₄)₂SO₄ was proposed by considering the contributing effects of solutes on the structure of water. The proposed equation satisfactorily described the solution system under investigation. The mean activity coefficients of H₂SO₄ and (NH₄)₂SO₄ in the solution system, H₂SO₄-(NH₄)₂SO₄-H₂O, at 298 K, were calculated by the McKay-Perring method using the water activities experimentally determined. It is believed that the calculated values for concentrated solutions of more than one sol-ute are sufficiently accurate since the mean activity coefficients of H₂SO₄ agree with the values measured by the emf method.     

Collaborative interactions between MEF-2 and Sp1 in muscle-specific gene regulation

We recently reported that previously activated T cells, irrespective of the nature of the first stimulus they encountered, are unable to respond to Staphylococcal enterotoxin B (SEB), nor to soluble anti-CD3 monoclonal antibody (mAb) presented by splenic antigen-presenting cells (APC). Such previously activated T cells are, however, fully capable of responding to plate-bound anti-CD3 plus splenic APC. These data suggest differential integration of the T-cell receptor (TCR) and co-stimulatory signalling pathways in naive versus antigen-experienced T cells. Consistent with this hypothesis, anti-CD28 mAb restores the proliferative capacity of resting ex vivo CD45RBlo CD4+ T cells (representing previously activated T cells) to both soluble anti-CD3 mAb and SEB. Interestingly, mAb-mediated engagement of cytotoxic T-lymphocyte antigen-4 (CTLA-4) completely negates the rescue effects mediated by anti-CD28 mAb in CD45RBlo cells. Nevertheless, the non-responsiveness of CD45RBlo CD4+ T cells cannot be reversed by anti-CTLA-4 Fab fragments, indicating that it is not related to negative regulatory effects of CTLA-4 engagement itself. Interestingly, the addition of interleukin-2 (IL-2) restores the proliferative capacity of CD45RBlo CD4+ T cells to SEB and soluble anti-CD3 mAb. Moreover, when rescued by IL-2, the cells are less susceptible to the negative regulatory effects of CTLA-4 engagement. Together, these findings suggest that the non-responsiveness of CD45RBlo CD4+ T cells to certain stimuli may be related to inadequate TCR signalling, primarily affecting IL-2 production.     

A high-resolution 1H NMR approach for structure determination of membrane peptides and proteins in non-deuterated detergent: application to mastoparan X solubilized in n-octylglucoside

Application of 1H 2D NMR methods to solubilized membrane proteins and peptides has up to now required the use of selectively deuterated detergents. The unavailability of any of the common biochemical detergents in deuterated form has therefore limited to some extent the scope of this approach. Here a 1H NMR method is described which allows structure determination of membrane peptides and small membrane proteins by 1H 2D NMR in any type of non-deuterated detergent. The approach is based on regioselective excitation of protein resonances with DANTE-Z or spin-pinging pulse trains. It is shown that regioselective excitation of the amide-aromatic region of solubilized membrane proteins and peptides leads to an almost complete suppression of the two orders of magnitude higher contribution of the protonated detergent to the 1H NMR spectrum. Consistently TOCSY, COSY and NOESY sequences incorporating such regioselective excitation in the F2 dimension yield protein 1H 2D NMR spectra of quality comparable to those obtained in deuterated detergents. Regioselective TOCSY and NOESY spectra display all through-bond and through-space correlations within amide-aromatic protons and between these protons and aliphatic and alpha-protons. Regioselective COSY spectra provide scalar coupling constants between amide and alpha-protons. Application of the method to the membrane-active peptide mastoparan X, solubilized in n-octylglucoside, yields complete sequence-specific assignments and extensive secondary structure-related spatial proximities and coupling constants.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cyclin D- and E-dependent kinases and the p57(KIP2) inhibitor: cooperative interactions in vivo

This study examines in vivo the role and functional interrelationships of components regulating exit from the G1 resting phase into the DNA synthetic (S) phase of the cell cycle. Our approach made use of several key experimental attributes of the developing mouse lens, namely its strong dependence on pRb in maintenance of the postmitotic state, the down-regulation of cyclins D and E and up-regulation of the p57(KIP2) inhibitor in the postmitotic lens fiber cell compartment, and the ability to target transgene expression to this compartment. These attributes provide an ideal in vivo context in which to examine the consequences of forced cyclin expression and/or of loss of p57(KIP2) inhibitor function in a cellular compartment that permits an accurate quantitation of cellular proliferation and apoptosis rates in situ. Here, we demonstrate that, despite substantial overlap in cyclin transgene expression levels, D-type and E cyclins exhibited clear functional differences in promoting entry into S phase. In general, forced expression of the D-type cyclins was more efficient than cyclin E in driving lens fiber cells into S phase. In the case of cyclins D1 and D2, ectopic proliferation required their enhanced nuclear localization through CDK4 coexpression. High nuclear levels of cyclin E and CDK2, while not sufficient to promote efficient exit from G1, did act synergistically with ectopic cyclin D/CDK4. The functional differences between D-type and E cyclins was most evident in the p57(KIP2)-deficient lens wherein cyclin D overexpression induced a rate of proliferation equivalent to that of the pRb null lens, while overexpression of cyclin E did not increase the rate of proliferation over that induced by the loss of p57(KIP2) function. These in vivo analyses provide strong biological support for the prevailing view that the antecedent actions of cyclin D/CDK4 act cooperatively with cyclin E/CDK2 and antagonistically with p57(KIP2) to regulate the G1/S transition in a cell type highly dependent upon pRb.     

Inter-helical interactions in the leucine zipper coiled coil dimer: pH and salt dependence of coupling energy between charged amino acids

We have investigated the physical nature of the observed coupling energy (Delta Delta DeltaGint) between the charged side-chains of the three inter-helical g<-->e' (i, i'+5) pairs (E<-->R, E<-->K, and E<-->E) in the leucine zipper coiled coil dimer. Circular dichroism (CD) spectroscopy measured the thermal stability of eight proteins derived from the basic region leucine zipper domain of chicken VBP, the mammalian TEF at seven pHs and three KCl concentrations. Data from these proteins were used to construct double mutant alanine thermodynamic cycles and determine coupling energies (Delta Delta DeltaGint) for the three g<-->e' pairs. The attractive E<-->R coupling energy of -0.6 kcal mol-1 at low salt decreases to -0.2 kcal mol-1 at high salt. The E<-->K coupling energy of -0.5 kcal mol-1 at low salt decreases to -0.1 kcal mol-1 at high salt. The repulsive E<-->E coupling energy of +0.8 kcal mol-1 at low salt drops to +0.4 at high salt. Reducing the pH to 2.2 halved the attractive coupling energy for the E<-->R and E<-->K pairs while abolishing the repulsion of the E<-->E pair. 13C NMR of a protein selectively labeled with [13Cdelta]glutamate that contained three E<-->R and one R<-->E pair identified four glutamates shifted upfield. We suggest that this is due to electronic perturbation of glutamates in inter-helical E<-->R interactions. Taken together, these data indicate that the E<-->R coupling energy of -0.5 kcal mol-1 at pH 7.4 and 150 mM KCl has an electrostatic component.     

High resolution 1H NMR spectroscopic studies of the metabolism and excretion of ampicillin in rats and amoxycillin in rats and man

High resolution proton nuclear magnetic resonance (1H NMR) spectroscopy has been used to investigate the metabolism and urinary excretion of the aminopenicillins, ampicillin and amoxycillin, in rats and of amoxycillin in man. 1H NMR resonances of the aminopenicillins, together with those for their 5R, 6R and 5S, 6R penicilloic acids and diketopiperazine metabolites were detected, assigned and quantified in urine samples with the aid of spin-echo NMR techniques. The dimer of amoxycillin was detected in rat urine for the first time together with novel drug-related resonances assigned to amoxycillin carbamate. Quantitative 1H NMR spectroscopic results were consistent with HPLC and microbiological data considering that only single measurements were recorded. Due to the short analysis time and simple sample preparation, NMR was particularly useful for studying the metabolism of the aminopenicillins for which sample degradation poses analytical problems. The non-invasive character of 1H NMR spectroscopic analysis of urine also provided unique information on a reversible reaction between amoxycillin and bicarbonate, an endogenous urinary metabolite.     

H2O2-treated actin: assembly and polymer interactions with cross-linking proteins

During inflammation, hydrogen peroxide, produced by polymorphonuclear leukocytes, provokes cell death mainly by disarranging filamentous (polymerized) actin (F-actin). To show the molecular mechanism(s) by which hydrogen peroxide could alter actin dynamics, we analyzed the ability of H2O2-treated actin samples to polymerize as well as the suitability of actin polymers (from oxidized monomers) to interact with cross-linking proteins. H2O2-treated monomeric (globular) actin (G-actin) shows an altered time course of polymerization. The increase in the lag phase and the lowering in both the polymerization rate and the polymerization extent have been evidenced. Furthermore, steady-state actin polymers, from oxidized monomers, are more fragmented than control polymers. This seems to be ascribable to the enhanced fragility of oxidized filaments rather than to the increase in the nucleation activity, which markedly falls. These facts; along with the unsuitability of actin polymers from oxidized monomers to interact with both filamin and alpha-actinin, suggest that hydrogen peroxide influences actin dynamics mainly by changing the F-actin structure. H2O2, via the oxidation of actin thiols (in particular, the sulfhydryl group of Cys-374), likely alters the actin C-terminus, influencing both subunit/subunit interactions and the spatial structure of the binding sites for cross-linking proteins in F-actin. We suggest that most of the effects of hydrogen peroxide on actin could be explained in the light of the "structural connectivity," demonstrated previously in actin.     

High-resolution mono- and multidimensional magic angle spinning 1H nuclear magnetic resonance of membrane peptides in nondeuterated lipid membranes and H2O

High-speed (14 kHz) solid-state magic angle spinning (MAS) 1H NMR has been applied to several membrane peptides incorporated into nondeuterated dilauroyl or dimyristoylphosphatidylcholine membranes suspended in H2O. It is shown that solvent suppression methods derived from solution NMR, such as presaturation or jump-return, can be used to reduce water resonance, even at relatively high water content. In addition, regioselective excitation of 1H peptide resonances promotes an efficient suppression of lipid resonances, even in cases where these are initially two orders of magnitude more intense. As a consequence, 1H MAS spectra of the peptide low-field region are obtained without interference from water and lipid signals. These display resonances from amide and other exchangeable 1H as well as from aromatic nonexchangeable 1H. The spectral resolution depends on the specific types of resonance and membrane peptide. For small amphiphilic or hydrophobic oligopeptides, resolution of most individual amide resonance is achieved, whereas for the transmembrane peptide gramicidin A, an unresolved amide spectrum is obtained. Partial resolution of aromatic 1H occurs in all cases. Multidimensional 1H-MAS spectra of membrane peptides can also be obtained by using water suppression and regioselective excitation. For gramicidin A, F2-regioselective 2D nuclear Overhauser effect spectroscopy (NOESY) spectra are dominated by intermolecular through-space connectivities between peptide aromatic or formyl 1H and lipid 1H. These appear to be compatible with the known structure and topography of the gramicidin pore. On the other hand, for the amphiphilic peptide leucine-enkephalin, F2-regioselective NOESY spectra mostly display cross-peaks originating from though-space proximities of amide or aromatic 1H with themselves and with aliphatic 1H. F3-regioselective 3D NOESY-NOESY spectra can be used to obtain through-space correlations within aliphatic 1H. Such intrapeptide proximities should allow determination of the conformation of the peptide in membranes. It is suggested that high-speed MAS multidimensional 1H NMR of peptides in nondeuterated membranes and in H2O can be used for studies of both peptide structure and lipid-peptide interactions.