Measurement of the Permanent Electric Dipole Moment of the Neutron.

We present the result of an experiment to measure the electric dipole moment (EDM) of the neutron at the Paul Scherrer Institute using Ramsey's method of separated oscillating magnetic fields with ultracold neutrons. Our measurement stands in the long history of EDM experiments probing physics violating time-reversal invariance. The salient features of this experiment were the use of a ^{199}Hg comagnetometer and an array of optically pumped cesium vapor magnetometers to cancel and correct for magnetic-field changes. The statistical analysis was performed on blinded datasets by two separate groups, while the estimation of systematic effects profited from an unprecedented knowledge of the magnetic field. The measured value of the neutron EDM is d_{n}=(0.0±1.1_{stat}±0.2_{sys})×10^{-26} e.cm.

Highly stable atomic vector magnetometer based on free spin precession.

We present a magnetometer based on optically pumped Cs atoms that measures the magnitude and direction of a 1 µT magnetic field. Multiple circularly polarized laser beams were used to probe the free spin precession of the Cs atoms. The design was optimized for long-time stability and achieves a scalar resolution better than 300 fT for integration times ranging from 80 ms to 1000 s. The best scalar resolution of less than 80 fT was reached with integration times of 1.6 to 6 s. We were able to measure the magnetic field direction with a resolution better than 10 µrad for integration times from 10 s up to 2000 s.

Observation of Gravitationally Induced Vertical Striation of Polarized Ultracold Neutrons by Spin-Echo Spectroscopy.

We describe a spin-echo method for ultracold neutrons (UCNs) confined in a precession chamber and exposed to a |B0|=1 µT magnetic field. We have demonstrated that the analysis of UCN spin-echo resonance signals in combination with knowledge of the ambient magnetic field provides an excellent method by which to reconstruct the energy spectrum of a confined ensemble of neutrons. The method takes advantage of the relative dephasing of spins arising from a gravitationally induced striation of stored UCNs of different energies, and also permits an improved determination of the vertical magnetic-field gradient with an exceptional accuracy of 1.1 pT/cm. This novel combination of a well-known nuclear resonance method and gravitationally induced vertical striation is unique in the realm of nuclear and particle physics and should prove to be invaluable for the assessment of systematic effects in precision experiments such as searches for an electric dipole moment of the neutron or the measurement of the neutron lifetime.

A Bell-Bloom experiment with polarization-modulated light of arbitrary duty cycle.

We report on a study of polarization-modulation experiments on the 4 → 3 hyperfine component of the D1 transition in Cs vapor contained in a paraffin-coated cell. The laser beam's polarization was switched between left- and right-circular polarization at a rate of 200 Hz. Variations of the transmitted light power were recorded while varying the amplitude of a transverse magnetic field. The power shows electromagnetically induced transparency (EIT) resonances when the atomic Larmor frequency matches a harmonic of the modulation frequency. We made a quantitative study of the resonance amplitudes with square-wave modulations of various duty cycles, and find an excellent agreement with recent algebraic model predictions.

Redox-mediated substrate recognition by Sdp1 defines a new group of tyrosine phosphatases.

Reactive oxygen species trigger cellular responses by activation of stress-responsive mitogen-activated protein kinase (MAPK) signalling pathways. Reversal of MAPK activation requires the transcriptional induction of specialized cysteine-based phosphatases that mediate MAPK dephosphorylation. Paradoxically, oxidative stresses generally inactivate cysteine-based phosphatases by thiol modification and thus could lead to sustained or uncontrolled MAPK activation. Here we describe how the stress-inducible MAPK phosphatase, Sdp1, presents an unusual solution to this apparent paradox by acquiring enhanced catalytic activity under oxidative conditions. Structural and biochemical evidence reveals that Sdp1 employs an intramolecular disulphide bridge and an invariant histidine side chain to selectively recognize a tyrosine-phosphorylated MAPK substrate. Optimal activity critically requires the disulphide bridge, and thus, to the best of our knowledge, Sdp1 is the first example of a cysteine-dependent phosphatase that couples oxidative stress with substrate recognition. We show that Sdp1, and its paralogue Msg5, have similar properties and belong to a new group of phosphatases unique to yeast and fungal taxa.

Visualization of dioxygen bound to copper during enzyme catalysis.

X-ray crystal structures of three species related to the oxidative half of the reaction of the copper-containing quinoprotein amine oxidase from Escherichia coli have been determined. Crystals were freeze-trapped either anaerobically or aerobically after exposure to substrate, and structures were determined to resolutions between 2.1 and 2.4 angstroms. The oxidation state of the quinone cofactor was investigated by single-crystal spectrophotometry. The structures reveal the site of bound dioxygen and the proton transfer pathways involved in oxygen reduction. The quinone cofactor is regenerated from the iminoquinone intermediate by hydrolysis involving Asp383, the catalytic base in the reductive half-reaction. Product aldehyde inhibits the hydrolysis, making release of product the rate-determining step of the reaction in the crystal.

Prolactin receptors in Rip-cre cells, but not in AgRP neurones, are involved in energy homeostasis.

Among its many functions, prolactin has been implicated in energy homeostasis, particularly during pregnancy and lactation. The arcuate nucleus is a key site in the regulation of energy balance. The present study aimed to examine whether arcuate nucleus neuronal populations involved in energy homeostasis are prolactin responsive and whether they can mediate the effects of prolactin on energy homeostasis. To determine whether Agrp neurones or Rip-Cre neurones are prolactin responsive, transgenic mice expressing the reporter td-tomato in Agrp neurones (td-tomato/Agrp-Cre) or Rip-Cre neurones (td-tomato/Rip-Cre) were treated with prolactin and perfused 45 minutes later. Brains were processed for double-labelled immunohistochemistry for pSTAT5, a marker of prolactin-induced intracellular signalling, and td-tomato. In addition, Agrp-Cre mice and Rip-Cre mice were crossed with mice in which the prolactin receptor gene (Prlr) was flanked with LoxP sites (Prlrlox/lox mice). The Prlrlox/lox construct was designed such that Cre-mediated recombination resulted in deletion of the Prlr and expression of green fluorescent protein (GFP) in its place. In td-tomato/Rip-Cre mice, prolactin-induced pSTAT5 was co-localised with td-tomato, indicating that there is a subpopulation of Rip-Cre neurones in the arcuate nucleus that respond to prolactin. Furthermore, mice with a specific deletion of Prlr in Rip-Cre neurones had lower body weights, increased oxygen consumption, increased running wheel activity and numerous cells in the arcuate nucleus had positive GFP staining indicating deletion of Prlr from Rip-Cre neurones. By contrast, no co-localisation of td-tomato and pSTAT5 was observed in td-tomato/Agrp-Cre mice after prolactin treatment. Moreover, Prlrlox/lox /Agrp-Cre mice had no positive GFP staining in the arcuate nucleus and did not differ in body weight compared to littermate controls. Overall, these results indicate that Rip-Cre neurones in the arcuate nucleus are responsive to prolactin and may play a role in the orexigenic effects of prolactin, whereas prolactin does not directly affect Agrp neurones.

Crystal structure of a quinoenzyme: copper amine oxidase of Escherichia coli at 2 A resolution.

BACKGROUND: Copper amine oxidases are a ubiquitous and novel group of quinoenzymes that catalyze the oxidative deamination of primary amines to the corresponding aldehydes, with concomitant reduction of molecular oxygen to hydrogen peroxide. The enzymes are dimers of identical 70-90 kDa subunits, each of which contains a single copper ion and a covalently bound cofactor formed by the post-translational modification of a tyrosine side chain to 2,4,5-trihydroxyphenylalanine quinone (TPQ). RESULTS: The crystal structure of amine oxidase from Escherichia coli has been determined in both an active and an inactive form. The only structural differences are in the active site, where differences in copper coordination geometry and in the position and interactions of the redox cofactor, TPQ, are observed. Each subunit of the mushroom-shaped dimer comprises four domains: a 440 amino acid C-terminal beta sandwich domain, which contains the active site and provides the dimer interface, and three smaller peripheral alpha/beta domains (D1-D3), each of about 100 amino acids. D2 and D3 show remarkable structural and sequence similarity to each other and are conserved throughout the quinoenzyme family. In contrast, D1 is absent from some amine oxidases. The active sites are well buried from solvent and lie some 35 A apart, connected by a pair of beta hairpin arms. CONCLUSIONS: The crystal structure of E. coli copper amine oxidase reveals a number of unexpected features and provides a basis for investigating the intriguing similarities and differences in catalytic mechanism of members of this enzyme family. In addition to the three conserved histidines that bind the copper, our studies identify a number of other conserved residues close to the active site, including a candidate for the catalytic base and a fourth conserved histidine which is involved in an interesting intersubunit interaction.

Structures of beta-ketoacyl-acyl carrier protein synthase I complexed with fatty acids elucidate its catalytic machinery.

BACKGROUND: beta-ketoacyl-acyl carrier protein synthase (KAS) I is vital for the construction of the unsaturated fatty acid carbon skeletons characterizing E. coli membrane lipids. The new carbon-carbon bonds are created by KAS I in a Claisen condensation performed in a three-step enzymatic reaction. KAS I belongs to the thiolase fold enzymes, of which structures are known for five other enzymes. RESULTS: Structures of the catalytic Cys-Ser KAS I mutant with covalently bound C10 and C12 acyl substrates have been determined to 2.40 and 1.85 A resolution, respectively. The KAS I dimer is not changed by the formation of the complexes but reveals an asymmetric binding of the two substrates bound to the dimer. A detailed model is proposed for the catalysis of KAS I. Of the two histidines required for decarboxylation, one donates a hydrogen bond to the malonyl thioester oxo group, and the other abstracts a proton from the leaving group. CONCLUSIONS: The same mechanism is proposed for KAS II, which also has a Cys-His-His active site triad. Comparison to the active site architectures of other thiolase fold enzymes carrying out a decarboxylation step suggests that chalcone synthase and KAS III with Cys-His-Asn triads use another mechanism in which both the histidine and the asparagine interact with the thioester oxo group. The acyl binding pockets of KAS I and KAS II are so similar that they alone cannot provide the basis for their differences in substrate specificity.

Electrochemical screening of self-assembling beta-sheet peptides using supported phospholipid monolayers.

In the context of the medical applications of beta-sheet self-assembling peptides, it is important to be able to predict their activity at the biological membrane level. A study of the interaction of four systematically varied 11-residue (P11-1, P11-2, P11-6 and P11-7) and one 13-residue (P13-1) designed beta-sheet self-assembling peptides with DOPC monolayers on a mercury electrode is reported in this paper. Experiments were carried out in 0.1 mol dm(-3) KCl electrolyte with added phosphate buffer (0.001 mol dm(-3)) at pH approximately 7.6. The capacity-potential curves of the coated electrode in the presence and absence of the different peptides were measured using out-of-phase ac voltammetry. The frequency dependence of the complex impedance of the coated electrode surfaces in the presence and absence of the peptides was estimated between 65,000 and 0.1 Hz at -0.4V versus Ag/AgCl 3.5 mol(-3) dm(-3) KCl. The monolayer permeabilising properties of the peptides were studied by following the reduction of Tl(I) to Tl(Hg) at the coated electrode. Of the five peptides studied, P11-2, P11-7 and P13-1 interact most strongly with the DOPC layer. P11-1 which has a polar primary structure shows no obvious interaction with the phospholipid but surprisingly, it permeabilises the phospholipid layer to Tl(+).

Comparison of two anti-Thy 1.1-abrin A-chain immunotoxins prepared with different cross-linking agents: antitumor effects, in vivo fate, and tumor cell mutants.

The A-chain of the plant toxin abrin was covalently linked to monoclonal anti-Thy 1.1 antibody (OX7) with the use of either N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) or 2-iminothiolane hydrochloride (2IT). The SPDP reagent generates a linkage containing a disulfide bond and an amide bond, whereas the 2IT reagent generates a linkage containing a disulfide bond and an amidinium bond. The two immunotoxins were powerfully and specifically toxic to Thy 1.1-expressing murine AKR-A lymphoma cells in vitro. Both reduced the rate of protein synthesis of the cells by 50% at a concentration of 10(-11) M. However, clonogenic assays revealed that about 1% of the AKR-A cells survived treatment with high concentrations of OX7-SPDP-abrin A, whereas only about 0.1% survived treatment with similar concentrations of OX7-2IT-abrin A. Several clones of the surviving cells were isolated. Of 11 clones of cells that had survived exposure to OX7-SPDP-abrin A, 10 were resistant to further treatment with OX7-SPDP-abrin A but had normal sensitivity to OX7-2IT-abrin A. These clones expressed moderate to high levels of the Thy 1.1 antigen and were fully sensitive to abrin. In contrast, all 10 clones of cells that had survived exposure to OX7-2IT-abrin A were substantially or entirely resistant to both immunotoxins. They expressed low to high levels of the Thy 1.1 antigen and were fully sensitive to abrin. The 2IT-linked immunotoxin was much more effective than the SPDP-linked immunotoxin at protecting nu/nu mice against the growth of AKR-A lymphoma cells in the peritoneal site. A single iv injection of 0.3 nmol OX7-2IT-abrin A eradicated at least 99.99% of the tumor cells, as judged from the extension in the median survival time of the animals, whereas OX7-SPDP-abrin A eradicated only about 99% of the cells. The tumors that developed in the animals that received OX7-2IT-abrin A were Thy 1.1-negative, whereas those in the recipients of OX7-SPDP-abrin A generally expressed normal levels of the Thy 1.1 antigen. The difference in antitumor activity of the immunotoxins was not due to differences in their in vivo fate, inasmuch as they were cleared from the bloodstream at an identical rate and broke down at the same rate to release free antibody.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of chemical deglycosylation of ricin A chain on the in vivo fate and cytotoxic activity of an immunotoxin composed of ricin A chain and anti-Thy 1.1 antibody.

The carbohydrate present on ricin A chain causes ricin A chain immunotoxins to be cleared rapidly in animals by the reticuloendothelial system. In an effort to overcome this problem we destroyed the carbohydrate on ricin A chain by treating it with a mixture of sodium metaperiodate and sodium cyanoborohydride and then linked the "deglycosylated" A chain to monoclonal anti-Thy 1.1 antibody. The deglycosylation procedure did not affect the ability of the A chain component of the immunotoxin to inhibit protein synthesis in a cell-free system or the capacity of the immunotoxin to inhibit protein synthesis in Thy-1.1 positive lymphoma cells in vitro. Immunotoxins prepared with deglycosylated A chain were cleared from the bloodstream of mice more slowly than native ricin A chain immunotoxins. The difference in the blood clearance rates of the two immunotoxins could be accounted for by a decreased entrapment of the deglycosylated ricin A chain immunotoxin by the liver. Both immunotoxins broke down in vivo with the appearance of free antibody in the bloodstream. The site of cleavage of the immunotoxin was possibly the liver because immunotoxins taken up by it rapidly became unreactive with antiricin but retained reactivity with anti-mouse immunoglobulin G suggesting that dissociation of the A chain from the antibody had occurred. The immunotoxins taken up by the liver were metabolized further and the acid insoluble radioactive metabolites gradually accumulated in the stomach, thyroid, and salivary gland. The deglycosylated ricin A chain immunotoxin should be a more effective antitumor agent in vivo because it is cleared from the blood more slowly and so has greater opportunity to localize within the tumor target.

Heparin-steroid conjugates: new angiogenesis inhibitors with antitumor activity in mice.

Inhibitors of angiogenesis hold potential in the treatment of cancer and other diseases where the disease is caused or maintained by the inappropriate growth of blood vessels. In the present study, a novel inhibitor of angiogenesis was synthesized by covalently linking a nonanticoagulating derivative of heparin, heparin adipic hydrazide (HAH), by an acid-labile bond to the antiangiogenic steroid, cortisol. The rationale was that the heparin derivative, which binds to sulfated polyanion receptors on endothelial cells, should concentrate the steroid on the surface of vascular endothelial cells. Endocytosis of the conjugate and decomposition of the acid-labile linkage inside lysosomes and other acidic intracellular compartments should then lead to release of the cortisol and expression of its antiproliferative activity. Analysis of the stability of HAH-cortisol showed that it was stable at pH 7.4 and broke down rapidly (t1/2 15 min) at pH 4.8 at 37 degrees C. Treatment of murine pulmonary capillary endothelial cells with HAH-cortisol at 10(-5) M (with respect to cortisol) suppressed their DNA synthesis by 50% and inhibited their migration into wounded areas of confluent monolayers. HAH-cortisol at 10(-4) M (with respect to cortisol) did not suppress the DNA synthesis of Lewis lung carcinoma cells. Daily i.p. injections of HAH-cortisol into mice bearing s.c. sponge implants retarded vascularization of the sponge, and injections directly into the sponge abolished vascularization for as long as the injections were continued. Daily i.v. injections of HAH-cortisol at doses causing no apparent toxicity retarded the growth of solid s.c. Lewis lung carcinomas in mice by up to 65%. In all of these assays, equivalent treatments with a mixture of the HAH plus cortisol was significantly less effective. The antiproliferative effect of HAH-cortisol on endothelial cells appeared independent of the glucocorticoid activity of the steroid since HAH conjugated to 5 beta-pregnane-3 alpha,17 alpha,21-triol-20-one, a steroid lacking glucocorticoid or mineralocorticoid activity, was even more effective at inhibiting DNA synthesis by murine pulmonary capillary endothelial cells than was HAH-cortisol. In conclusion, HAH-cortisol represents the prototype of a new class of angiogenesis inhibitors for the treatment of cancer and other angiogenic diseases.

Improved antitumor effects of immunotoxins prepared with deglycosylated ricin A-chain and hindered disulfide linkages.

A monoclonal anti-Thy-1.1 antibody (OX7) was coupled to either native or chemically deglycosylated ricin A-chain (dgA) using one of two different cross-linking agents. One cross-linker, N-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pyridyldithio)tolu ene (SMPT), generates a sterically hindered disulfide bond which is relatively resistant to reduction, whereas the other, 2-iminothiolane hydrochloride, generates an unhindered disulfide bond with greater lability. A two-compartment pharmacokinetic model was used to analyze the blood levels of each immunotoxin and its breakdown product (free antibody) after i.v. injection into mice. Immunotoxins prepared with SMPT broke down in vivo 6.3-fold more slowly than those prepared with 2-iminothiolane hydrochloride, and immunotoxins containing native A-chain were cleared 2- to 3-fold more rapidly from the bloodstream than those containing dgA. As a result, 24 h after injection, 16% of the OX7-SMPT-dgA remained in the blood as compared with 0.4 to 2.5% of the other immunotoxins. Immunotoxins prepared with dgA were about 3-fold more toxic to mice than those prepared with native A-chain, whereas immunotoxins prepared with SMPT were only slightly more toxic than those prepared with 2-iminothiolane hydrochloride. When equivalent toxic doses of the immunotoxins were administered i.v. to mice which had been given injections of Thy-1.1+ AKR-A/2 lymphoma cells, the OX7-SMPT-dgA gave the best antitumor effect. A dose equivalent to one-seventh of the median lethal dose extended the survival time of the animals by the extent expected if 99.999% of the tumor cells had been eradicated. Furthermore, the tumors that did develop in the mice treated with OX7-SMPT-dgA were mutants which were resistant to all the immunotoxins. Some of the mutants were deficient in Thy-1.1 whereas others were not. In conclusion, both the use of the SMPT cross-linker and deglycosylation of the A-chain significantly improve the therapeutic index of the immunotoxins in AKR-A/2 tumor-bearing mice.

Evaluation of ricin A chain-containing immunotoxins directed against CD19 and CD22 antigens on normal and malignant human B-cells as potential reagents for in vivo therapy.

Ricin A chain-containing immunotoxins (IT-As) specific for the human B-cell antigens, CD22 and CD19, were constructed using the monoclonal antibodies, HD6 and HD37, respectively. IT-As were prepared by coupling intact antibodies, F(ab')2, or Fab' fragments to native or chemically deglycosylated ricin A chain. The IT-As were then evaluated for cytotoxicity to normal and neoplastic human B-cells in vitro with the major objective of appraising their suitability for in vivo therapy of human B-cell tumors. The IT-As prepared with both the HD6 and HD37 antibodies were specifically toxic to normal B-cells and to most of the neoplastic B-cell lines tested. However, the IT-As prepared from HD6 were generally more potent than those prepared from HD37. On Daudi cells, to which the two antibodies bound in similar numbers and with similar affinities, IT-As prepared with intact HD6 antibody or its Fab' fragment were 10-fold and 1.5- to 4-fold more potent, respectively, than the corresponding HD37 IT-As. The IT-As constructed from intact HD6 antibody and native or deglycosylated A chain reduced protein synthesis in Daudi cells by 50% at a concentration of 1.2 X 10(-11) M indicating that they were only 5-fold less toxic to the cells than ricin itself. Intact HD37 IT-As produced equivalent inhibition of protein synthesis at 1.5 X 10(-10) M. With both antibodies, IT-As constructed from the Fab' fragments were 10- to 20-fold less potent than their intact antibody counterparts. Different neoplastic B-cell lines varied in sensitivity to the IT-As. In most cases, their sensitivity correlated with the levels of CD19 and CD22 antigens expressed. Neither HD6 nor HD37 IT-As affected the ability of normal human bone marrow cells to form granulocyte-macrophage colony-forming units in soft agar, suggesting that both antigens are absent from these progenitor cells. Examination of sections of frozen human tissues using immunoperoxidase staining procedures indicated that the antibodies did not bind to a panel of normal tissues lacking B-lymphocytes. These results suggest that HD6 and HD37 IT-As are candidates for in vivo therapy in humans with certain B-cell tumors. However, HD6 IT-As are more potent, reduce protein synthesis more completely, and hence appear to be the ITs of choice for treating tumors expressing the CD22 antigen.

New coupling agents for the synthesis of immunotoxins containing a hindered disulfide bond with improved stability in vivo.

Two new coupling agents were synthesized for making immunotoxins containing disulfide bonds with improved stability in vivo: sodium S-4-succinimidyloxycarbonyl-alpha-methyl benzyl thiosulfate (SMBT) and 4-succinimidyloxycarbonyl-alpha-methyl-alpha(2-pyridyldithio)tolue ne (SMPT). Both reagents generate the same hindered disulfide linkage in which a methyl group and a benzene ring are attached to the carbon atom adjacent to the disulfide bond and protect it from attack by thiolate anions. An immunotoxin consisting of monoclonal anti-Thy-1.1 antibody (OX7) linked by means of the SMPT reagent to chemically deglycosylated ricin A-chain had better stability in vivo than an immunotoxin prepared with 2-iminothiolane hydrochloride (2IT) which generates an unhindered disulfide linkage. About 48 h after i.v. injection into mice, one-half of the SMPT-linked immunotoxin present in the blood was in intact form and one-half as released free antibody, whereas equivalent breakdown of the 2IT-linked immunotoxin was seen at about 8 h after injection. Consequently, the blood levels of the SMPT-linked immunotoxin remained higher than those of the 2IT-linked immunotoxin despite loss of immunotoxin from the blood by other mechanisms. Forty-eight h after injection, 10% of the injected dose of the SMPT-linked immunotoxin remained in the bloodstream as compared with only 1.5% of the 2IT-linked immunotoxin. The ability of immunotoxins prepared with the new reagents to inhibit protein synthesis by Thy-1.1-expressing AKR-A/2 lymphoma cells in vitro was identical to that of immunotoxins prepared with 2IT or N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP). Clonogenic assays showed that fewer than 0.01% of AKR-A/2 cells survived exposure to high concentrations of OX7-abrin A-chain immunotoxins prepared with SMBT, 2IT, or SPDP. Twelve clones of cells which had survived treatment with the SMBT-linked immunotoxin were isolated. None of the clones was selectively resistant to the SMBT-linked immunotoxin when retested in cytotoxicity assays. In conclusion, immunotoxins prepared with the new coupling agents should have improved antitumor activity in vivo because they are longer lived and do not break down so readily to release free antibody which could compete for the target antigens.

Osteoprotective Effects of Estrogen in the Maxillary Bone Depend on ERα.

Estrogen deficiency results in disruption of maxillary alveolar bone microarchitecture. Most of the actions of estrogen in long bones occur via estrogen receptor α (ERα). However, the function of ERα in the maxillary bone has not been defined. We aimed to investigate the role and underlying mechanisms of ERα in the physiological and mechanically induced alveolar bone remodeling in female and male mice. Wild-type (WT) and ERα(-/-) (ERKOα) mice were subjected to mechanically stimulated bone remodeling by inducing orthodontic tooth movement (OTM). The maxillary bone was analyzed using histomorphometric analysis, micro-computed tomography, quantitative polymerase chain reaction, and energy-dispersive spectroscopy. Bone marrow cells (BMCs) from WT and ERKOα mice were tested for their capacity to differentiate into osteoblasts and osteoclasts. Both male and female ERKOα mice exhibited marked reduction of alveolar bone mass and increased OTM. This response was associated with an increased number of osteoclasts and reduced number of apoptotic cells and osteoblasts in the periodontium and alveolar bone. Consistently, ERKOα mice exhibited lower levels of calcium in bone and increased expression of IL-33 (interleukin-33), TNF-α (tumor necrosis factor α), and IL-1ß (interleukin-1ß) and decreased expression of dentin matrix acidic phosphoprotein and alkaline phosphatase in periodontal tissues. Moreover, the differentiation of osteoclasts and osteoblasts in vitro was significantly higher in BMCs obtained from ERKOα. ERα is required to maintain the microarchitecture of maxillary alveolar bone. This process is linked to bone cell differentiation and apoptosis, as well as local production of inflammatory molecules such as IL-33, TNF-α, and IL-1ß.

Divalent metal ions induce conformational change in pure, human wild-type p53 tumor suppressor protein.

The ability of wild-type and not mutant p53 to exert antiproliferative effects on normal cells may be related to a difference in the conformational state of the protein. We have used pure, human wild-type p53 and a panel of monoclonal antibodies whose epitopes map throughout the protein to assess whether divalent metal ions affect the conformation of p53. Our results show that the presence of Zn2+ ions at physiological concentrations, directly reduced or blocked accessibility of epitopes on pure wild-type p53, an effect which was reversed by chelating agents. Loss of epitope reactivity was maximal between the protein mid-region and C-terminus. Analytical sucrose density gradient ultracentrifugation studies also confirmed that Zn(2+)-induced conformational changes partially affected the pattern of p53 oligomerisation. The observed binding of pure p53 to a sequence-specific DNA motif was unaffected by the presence of added Zn2+ ions or metal chelating agents.

Association of spin-labelled cardiolipin with dimyristoylphosphatidylcholine-substituted bovine heart cytochrome c oxidase. A generalized specificity increase rather than highly specific binding sites.

The endogeneous lipid of bovine heart cytochrome c oxidase has been replaced by dimyristoylphosphatidylcholine using cholate-mediated exchange. The lipid-substituted preparation contained less than 1 mole cardiolipin per mole enzyme and possessed full oxidative activity. The association of spin-labelled cardiolipin with such lipid-substituted cytochrome oxidase preparations has been assayed using ESR spectroscopy. An average relative association constant 5.4-times that for phosphatidylcholine is obtained for cardiolipin. Measurements on preparations with increasing contents of unlabelled cardiolipin, introduced during lipid exchange, reveal that this selectivity corresponds to a generalized increase in specificity for all lipid association sites on the protein.

Cooperativity of the phase transition in single- and multibilayer lipid vesicles.

The effect of membrane morphology on the cooperativity of the ordered-fluid, lipid phase transition has been investigated by comparing the transition widths in extended, multibilayer dispersons of dimyristoyl phosphatidyl-choline, and also of dipalmitoyl phosphatidylcholine, with those in the small, single-bilayer vesicles obtained by sonication. The electron spin resonance spectra of three different spin-labelled probes, 2,2,6,6-tetramethylpiperdine-N-oxyl, phosphatidylcholine and stearic acid, and also 90 degrees light scattering and optical turbidity measurements were used as indicators of the phase transition. In all cases the transition was broader in the single-bilayer vesicles than in the multibilayer dispersions, corresponding to a decreased cooperativity on going to the small vesicles. Comparison of the light scattering properties of centrifuged and uncentrifuged, sonicated vesicles suggests that these are particularly sensitive to the presence of intermediate-size particles, and thus the spin label measurements are likely to give a more reliable measure of the degree of cooperativity of the small, single-bilayer vesicles. Application of the Zimm and Bragg theory ((1959) J. Chem. Phys. 31, 526-535) of cooperative transitions to the two-dimensional bilayer system shows that the size of the cooperative unit, 1/square root sigma, is a measure of the mean number of molecules per perimeter molecule, in a given region of ordered or fluid lipid at the centre of the transition. From this result it is found that it is the vesicle size which limits the cooperativity of the transition in the small, single-bilayer vesicles. The implications for the effect of membrane structure and morphology on the cooperativity of phase transitions in biological membranes, and for the possibility of achieving lateral communication in the plane of the membrane, are discussed.