nH Values Research Articles

X-Ray, Optical, and Infrared Imaging and Spectral Properties of the 1 M[CLC]s[/CLC] Chandra Deep Field North Sources

We present the optical, near-infrared, submillimeter, and radio follow-up catalog of the X-ray–selected sources from an ≈1 Ms Chandra observation of the Hubble Deep Field North region. We have B, V, R, I, and z' magnitudes for the 370 X-ray point sources, HK' magnitudes for 276, and spectroscopic redshifts for 182. We present high-quality spectra for 175 of these. The redshift distribution shows indications of structures at z = 0.843 and 1.0175 (also detected in optical surveys), which could account for a part of the field-to-field variation seen in the X-ray number counts; however, these structures do not dominate the number of X-ray sources in the sample and hence should not strongly affect the redshift distribution. All of the X-ray sources with z > 1.6 are either broad-line active galactic nuclei (AGNs) or have narrow Lyα and/or C III] 1909 A emission; none of the known z > 1.6 absorption-line galaxies in the field are detected individually in X-rays. We estimate photometric redshifts for the sources with (B-I) > 1.5 (bluer than this and it is hard to distinguish between low-redshift irregulars and luminous high-redshift AGNs) and find agreement (most are within 25%) with the available spectroscopic redshifts. The majority of the galaxies in both the 2–8 keV (hard) and 0.5–2 keV (soft) samples have absolute magnitudes comparable to or more luminous than M = -22. The flux contributions separated into unit bins of redshift show that the z < 1 spectroscopically identified sources already contribute about one-third of the total flux in both the hard and soft bands. Thus, major accretion onto supermassive black holes has occurred since the universe was half its present age. We find from ratios of the X-ray counts that the X-ray spectra are well described by absorption of an intrinsic Γ = 1.8 power law, with NH values ranging from about 1021 cm-2 to 5 × 1023 cm-2. We find very little evolution in the maximum rest-frame opacity-corrected and K-corrected 2–8 keV X-ray luminosities with decreasing redshift until z 0.5, where the volume becomes too small to probe effectively very high luminosity sources. We estimate that the Chandra sources that produce 87% of the HEAO A X-ray background (XRB) at 3 keV produce 57% at 20 keV, provided that at high energies the spectral shape of the sources continues to be well described by a Γ = 1.8 power law. However, when the Chandra contributions are renormalized to the BeppoSAX XRB at 3 keV, the shape matches fairly well the observed XRB at both energies. Thus, whether a substantial population of as yet undetected Compton-thick sources or some change in the spectral shape of the current sources from the simple power-law dependence is required to completely resolve the XRB above 10 keV depends critically on how the currently discrepant XRB measurements in the 1-10 keV energy range tie together with the higher energy XRB.

Temperature Effects on the Allosteric Transition of ATP Sulfurylase from Penicillium chrysogenum

The effects of temperature on the initial velocity kinetics of allosteric ATP sulfurylase from Penicillium chrysogenum were measured. The experiments were prompted by the structural similarity between the C-terminal regulatory domain of fungal ATP sulfurylase and fungal APS kinase, a homodimer that undergoes a temperature-dependent, reversible dissociation of subunits over a narrow temperature range. Wild-type ATP sulfurylase yielded hyperbolic velocity curves between 18 and 30°C. Increasing the assay temperature above 30°C at a constant pH of 8.0 increased the cooperativity of the velocity curves. Hill coefficients (nH) up to 1.8 were observed at 42°C. The bireactant kinetics at 42°C were the same as those observed at 30°C in the presence of PAPS, the allosteric inhibitor. In contrast, yeast ATP sulfurylase yielded hyperbolic plots at 42°C. The P. chrysogenum mutant enzyme, C509S, which is intrinsically cooperative (nH = 1.8) at 30°C, became more cooperative as the temperature was increased yielding nH values up to 2.9 at 42°C. As the temperature was decreased, the cooperativity of C509S decreased; nH was 1.0 at 18°C. The cumulative results indicate that increasing the temperature increases the allosteric constant, L, i.e., promotes a shift in the base-level distribution of enzyme molecules from the high MgATP affinity R state toward the low MgATP affinity T state. As a result, the enzyme displays a true “temperature optimum” at subsaturating MgATP. The reversible temperature-dependent transitions of fungal ATP sulfurylase and APS kinase may play a role in energy conservation at high temperatures where the organism can survive but not grow optimally.

Joint features of pressure effects and specific heat of RBa 2Cu 3Ox at distinct nh values

Investigations of specific heat and T c under pressure are presented for YBa 2Cu 3O x (Y123), NdBa 2Cu 3O x (Nd123) and Y 1−yCa yBa 2Cu 3O x (YCa123) for various oxygen contents. In Y123, at the maximum of the specific-heat jump ΔC/T c the pressure dependence of the transition temperature dT c/dp shows a zero crossing. Inversely, at the maximum of dT c/dp the jump in the specific heat becomes minimal. With Ca doping these joint features shift to lower oxygen contents x, whereas Nd123 causes a shift into the opposite direction. Considering the hole doping by Ca and the larger chain fragmentation in Nd123, we rescaled our results with respect to the hole content n h in the CuO 2 planes. In this plot features coalesce in two fixed hole concentrations, n h≈0.11 and n h0.17, irrespective of the substitution and doping. The maximum in ΔC/T c may be related to the appearance of a quantum critical point due to the disappearance of a pseudogap for hole contents above n h≈0.17. The features at n h≈0.11 point to a drastic change in system properties at this hole content that may be connected to various anomalies found in other properties reported in the literature.

Selective hydration of alkylammonium ions in nitrobenzene

In the nitrobenzene (NB)/water system, primary to tertiary ammonium ions with Me, Et and n-Bu groups were distributed with dipicrylaminate (DPA−) and tetraphenylborate (TPB−), and the number of water molecules being co-extracted to NB by an alkylammonium ion (RmNH4−m+) was measured by the Karl Fischer method. In the use of TPB− as the counter ion, the numbers of co-extracted water molecules, mainly for primary and secondary alkylammonium ions, were somewhat smaller than those in the use of DPA−, showing a possible influence of ion-pair formation between RmNH4−m+ and TPB− in NB. Conductivity measurements were then performed to determined the association constants of the RmNH4−m+ ions with TPB− ions as well as DPA− in ‘‘water-saturated ’’ NB, and the concentrations of free RmNH4−m+ ions were evaluated. By assuming that the ion pairs of RmNH4−m+ would have no water molecules in NB, the ‘‘ true’’ hydration numbers (nh) of the RmNH4−m+ ions in NB were successfully obtained. The nh values of the RmNH4−m+ ions, being little affected by the alkyl chain length, were found to decrease with heightening the class of the RmNH4−m+ ion: nh=1.64, 1.04 and 0.66, respectively, for the primary, secondary and tertiary ammonium ions. It was suggested that water molecules interact directly with hydrogen atom(s) bound to the central nitrogen atom.

Synthesis and pharmacology of site-specific cocaine abuse treatment agents: 2-(aminomethyl)-3-phenylbicyclo[2.2.2]- and -[2.2.1]alkane dopamine uptake inhibitors.

As part of a program to develop site-specific medications for cocaine abuse, a series of 2-(aminomethyl)-3-phenylbicyclo[2.2.2]- and -[2.2.1]alkane derivatives was synthesized and tested for inhibitory potency in [3H]WIN 35,428 binding and [3H]dopamine uptake assays using rat striatal tissue. Selected compounds were tested for their ability to substitute for cocaine in rat drug discrimination tests. Synthesis was accomplished by a series of Diels-Alder reactions, using cis- and trans-cinnamic acid derivatives (nitrile, acid, acid chloride) with cyclohexadiene and cyclopentadiene. Standard manipulations produced the aminomethyl side chain. Many of the compounds bound with high affinity (median IC50 = 223 nM) to the cocaine binding site as marked by [3H]WIN 35,428. Potency in the binding assay was strongly enhanced by chlorine atoms in the 3- and/or 4-position on the aromatic ring and was little affected by corresponding methoxy groups. In the [2.2.2] series there was little difference in potency between cis and trans compounds or between N, N-dimethylamines and primary amines. In the [2.2.1] series the trans exo compounds tended to be least potent against binding, whereas the cis exo compounds were the most potent (4-Cl cis exo: IC50 = 7.7 nM, 27-fold more potent than 4-Cl trans-exo). Although the potencies of the bicyclic derivatives in the binding and uptake assays were highly correlated, some of the compounds were 5-7-fold less potent at inhibiting [3H]dopamine uptake than [3H]WIN 35,428 binding (for comparison, cocaine has a lower discrimination ratio (DR) of 2.5). The DR values were higher for almost all primary amines and for the trans-[2.2.2] series as compared to the cis-[2.2.2]. Most of the compounds had Hill coefficients approaching unity, except for the [2. 2.2] 3,4-dichloro derivatives, which all had nH values of about 2.0. Two of the compounds were shown to fully substitute for cocaine in drug discrimination tests in rats, and one had a very long duration of action.

High‐Energy Spectra of Active Galactic Nuclei. II. Absorption in Seyfert Galaxies

Absorption by cold material in a large sample of active galaxies has been analyzed in order to study statistically the behavior of absorbed sources. The analysis indicates that on the basis of the column density alone, sources can be divided into low-absorption ([NH/NHGal] ? 50) and high-absorption ([NH/NHGal] ? 50) objects. While the second group consists mostly of narrow emission line galaxies (Seyfert galaxies of type 1.9-2), the first group is less homogenous, being formed by a mixture of broad and narrow emission line objects (Seyfert 1-2 galaxies). A study of the distribution of the column density values by means of bootstrap analysis confirms the reality of this effect. One group consisting of optically selected objects is well explained within the unified theory as nuclei obscured by a molecular torus. The second group made up of X-ray- and IRAS-selected objects is more difficult to define: in these sources the absorption is underestimated owing to difficulties (1) in fitting complex absorption spectra or (2) in measuring NH values in Compton-thick sources or the absorption has a different origin than in the torus. Possible correlations of absorption with X-ray luminosity, axial ratio, and Balmer decrement have also been investigated. Previous suggestions that lower luminosity AGNs tend to be more highly absorbed than those with higher luminosity are not confirmed by the present data; neither is any evidence for a correlation of NH with axial ratio (b/a) found except for a preference of Seyfert 1-1.5 galaxies to be in face-on galaxies. While some sources (Seyfert 1-1.5 galaxies and low-absorption objects) have X-ray absorption compatible with Balmer decrement, high-absorption objects have column densities much higher than predicted from optical observations. These results are in agreement with the unified theory since the torus parameters are expected to be independent of luminosity, its orientation should be random with respect to the host galaxy, and its location should be in between the broad- and narrow-line regions. A study of the NH variability indicates that in a large fraction (70%) of the sources for which the analysis could be done, NH varies on timescales from months to years. In Seyfert 1-1.5 galaxies, the variability is associated with a region in or near the broad-line region and is explained in terms of partial covering and/or warm absorption models. In Seyfert 2 galaxies, the only variability observed is that associated with narrow emission line galaxies. The study of the column density distributions indicates that Seyfert 1-1.5 galaxies are characterized by NH = 18+9?7 ? 1021 atoms cm-2. Seyfert 1.9-2 galaxies have instead NH = 96+54?35 ? 1021 atoms cm-2 and a larger dispersion; if this group is divided into low- and high-absorption objects, NH = 14.5+7.2?5.3 ? 1021 atoms cm-2 and NH = 132.8+80.1?52.6 ? 1021 atoms cm-2, respectively, are obtained. The observed dispersion in each group is consistent with being entirely due to column density variability.

Ritter reactions. XII. Reappraisal of the reactivity of methyl Schiff bases with dimethyl acetylenedicarboxylate

The reaction of the methyl Schiff base compound 2 with dimethyl acetylenedicarboxylate (DMAD) has been reinvestigated in order to determine whether it is initiated through the imine form 2 or the enamine tautomer 3. The structure of the reported 1:1 adduct is reassigned as 6 following elucidation by a combination of NMR techniques including natural abundance gradient enhanced 1H- 15N HMBC and 1D HSQC, as well as 1H, 13C, DEPT, DQF-COSY, NOESY, 1H- 13C HMQC and HMBC. Similar experiments, plus 13C- 13C INADEQUATE data, for the 1:1 adduct of imine 10 and DMAD require its reassignment as structure 15. 3 J NH values were used as an indicator of the likely geometries of these products. In both cases, the products are the eventual outcome of initial attack on DMAD by the nitrogen of the imine form.

Effects of extracellular pH on agonism and antagonism at a recombinant P2X2 receptor.

1. Under voltage-clamp conditions, the activity of agonists and antagonists at a recombinant P2X2 receptor expressed in Xenopus oocytes was examined at different levels of extracellular pH (pHe). 2. In normal Ringer (Mg2+ ions absent), the amplitude of submaximal inward currents to ATP was increased by progressively lowering pHe (8.0-5.5). ATP-responses reached a maximum at pH 6.5 with a 5 fold increase in ATP-affinity; the apparent pKa was 7.05 +/- 0.05. 3. Receptor affinity for ATP was lowered when extracellular Ca2+ ions were replaced with equimolar Mg2+ ions. However, the amplitude of the ATP-responses was still enhanced under acidic conditions, reaching maximal activity at pH 6.5 with a 5 fold increase in ATP-affinity; the apparent pKa was 7.35 +/- 0.05. 4. ATP species present in the superfusate (for the above ionic conditions and pH levels) were calculated to determine the forms of ATP which activate P2X2 receptors: possible candidates include HATP, CaHATP and MgHATP. However, levels of these protonated species increase below pH 6.5, suggesting that receptor protonation rather than agonist protonation is more important. 5. The potency order for agonists of P2X2 receptors was: ATP> 2-MeS-ATP ATPgammaS> ATPalphaS> >CTP >BzATP, while other nucleotides were inactive. EC50 and nH values for full agonists were determined at pH 7.4 and re-examined at pH 6.5. Extracellular acidification increased the affinity by approximately 5 fold for full agonists (ATP, 2-MeSATP, ATPyS and ATP alpha S), without altering the potency order. 6. The potency order for antagonists at P2X2 receptors was: Reactive blue-2 >trinitrophenol-ATP > or = Palatine fast black > or = Coomassie brilliant blue > or = PPADS>suramin (at pH 7.4). IC50 values and slopes of the inhibition curves were re-examined at different pH levels. Only blockade by suramin was affected significantly by extracellular acidification (IC50 values: 10.4 +/- 2 microM, at pH 7.4; 78 +/- 5 nM, at pH 6.5; 30 +/-6 nM, at pH 5.5). 7 In summary, a lowered pHe enhanced the activity of all agonists at P2X2 receptors but, with the exception of suramin, not antagonists. Since a lowered pHe is also known to enhance agonist activity at P2x receptors on sensory neurones containing P2X2 transcripts, the sensitization by metabolic acidosis of native P2x receptors containing P2X2 subunits may have a significant effect on purinergic cell-to-cell signalling.

Characterization of NK3 receptors in rabbit isolated iris sphincter muscle.

1. Tachykinin NK3 receptors were characterized in the rabbit isolated iris sphincter muscle by use of autoradiography and in vitro functional studies. 2. [125I]-[MePhe7]-neurokinin B (NKB) (1nM), a selective NK3 receptor agonist, specifically labelled a population of NK3 receptors that were uniformly distributed throughout the rabbit iris sphincter muscle. This labelling was inhibited by unlabelled [MePhe7]-NKB (1 microM) but not by the NK1 receptor antagonist CP 99994 (1 microM). 3. In the presence of CP 99994 (1 microM), the selective NK3 receptor agonists senktide (n = 14) and [Pro7]-NKB (n = 4), and the natural preferred ligand for the NK3 receptor, NKB (n = 8), were potent contractile agents in the rabbit iris sphincter muscle. They all produced monophasic concentration-effect curves with pD2 values of 9.53 +/- 0.08, 8.56 +/- 0.09 and 9.75 +/- 0.09, and nH values of 0.93 +/- 0.03, 1.53 +/- 0.17 and 0.76 +/- 0.06, respectively. [MePhe7]-NKB (n = 12) was also a potent agonist, but produced shallow concentration-effect curves which appeared biphasic (nH = 0.45 +/- 0.04). 4. Contractile responses to senktide were surmountably antagonized in a concentration-dependent manner by the selective non-peptide NK3 receptor antagonist, SR 142801 (3-30 nM; pA2 = 8.9; slope = 0.99) and the non-peptide NK2/NK3 receptor antagonist, SR 48968 (3-30 microM; pA2 = 6.1; slope = 1.5). These pA2 values were consistent with functional rabbit NK3 receptors more closely resembling guinea-pig and human NK3 receptors, than rat NK3 receptors. SR 142801 (10-100 nM) and SR 48968 (3 and 30 microM) inhibited responses to low (< or = 1 nM) but not higher (> 1 nM) concentrations of [MePhe7]-NKB, and concentration-effect curves to [MePhe7]-NKb became steeper and monophasic in the presence of either antagonist. 5. SR 142801 (3-30 nM) and SR 48968 (3-30 microM) also surmountably antagonized concentration-effect curves to [Pro7]-NKB and NKB, although results were more difficult to interpret, since the relationship between log concentration-ratios and the concentration of antagonist used did not adhere to the Schild equation. However, analysis of data with the lowest concentration of SR 142801 (3 nM) tested against NKB, and SR 48968 (3 microM) tested against [Pro7]-NKB and NKB, yielded apparent pA2 estimates of 9.3, 6.8 and 6.4, respectively, consistent with blockade of NK3 receptors. 6. SR 142801 (100 nM) had no effect on contractions induced by transmural nerve stimulation (2 Hz, 0.3 ms, 20 V for 30 s), whereas CP 99994 (1 microM) abolished these responses. 7. Phenoxybenzamine pretreatment (20 microM, 10 min) markedly reduced maximum responses to [MePhe7]-NKB (from 101 +/- 6.2% to 38 +/- 9.5% reference contraction, n = 4) and induced a marked (10 fold) rightward shift in the concentration-effect curve. The residual responses to [MePhe7]-NKB after phenoxybenzamine pretreatment were unaffected by 1 microM CP 99994 (maximum response = 41 +/- 9.4%, n = 4). 8. These results demonstrate autoradiographically and functionally, the presence of NK3 receptors in rabbit iris sphincter muscle that mediate contractile responses to NK3 receptor agonists, but not to sensory trigeminal nerve simulation. The present data with senktide and selective NK3 receptor antagonists suggest that functional rabbit NK3 receptors more closely resemble human and guinea-pig NK3 receptors than rat NK3 receptors. However, the pharmacological profiles of [MePhe7]-NKB, SR 142801 and SR 48968 suggest the presence of an 'atypical' NK3 receptor or a heterogeneous population of NK3 receptors in this tissue.

NMR study of proton location in strongly hydrogen bonded complexes of pyridine as influenced by solvent polarity

1H, 15N and 13C NMR spectra of H-bonded complexes of 15N-enriched pyridine (Y) with various strong proton donors (XH) were obtained in a range of solvents with different dielectric constant at low temperature. Chemical shifts and nuclear magnetic relaxation times, T 1, were measured. A procedure for evaluating the mean NH distance in the complexes using T 1 values for 15N and 13C nuclei treated as determined by dipole-dipole coupling with nearest protons is proposed. The results point to a gradual shift of the average proton position towards the N atom, with increasing the XH acidity. For a given complex, the NH distance falls with the ε dielectric constant of a solvent, linear correlation between rNH and the reactive field factor, F=( ε-1)/(2 ε+1), being observed. The analogous correlation was found for the Δδ values. For interpretation of the effect, an anharmonic two-dimensional model of H-bond in connection with the Onsager-Butcher potential was used. It was found that the reactive field of a solvent causes the NH bond to shorten due to the specific feature of the dipole moment function common for nearly all XH…Y and X −…HY + complexes ( ∂μ/ ∂R Y-H<0). With the required parameters taken from IR spectra, this model gives a reasonable coincidence with the experimental Ar NH values.

Probing [CLC]Lyα[/CLC] Absorbers with Double Lines of Sight

Study of Lyα absorption lines in the spectra of double line-of-sight (DLOS) quasars holds the promise of diagnosing the nature of the structures that give rise to Lyα absorption. Based on simulations of DLOS with various separations through a single population of absorbers, four tests have been designed to diagnose absorber mass distributions (smoothly varying with radius or irregular), geometries (spherical or disk/slab-like), and kinematics (isotropic or systematic velocities). Applying the tests to existing data at z ~ 2 we find the following. (1) The observed neutral hydrogen column density (NH) distributions of Lyα lines coincident to both LOSs are consistent with a smooth mass distribution. (2) Observed large anticoincident NH values are not consistent with a single population of smooth spherical absorbers, which should exhibit sharp cutoffs at small NH in the NH distribution. (3) There is marginal evidence that the observed rms value of velocity differences between coincident lines increases with DLOS separation, as is expected for disk/slab-like absorbers that have systematic velocity fields. (4) The observed velocity dispersion along a single LOS is small compared to the rms difference between widely separated LOS, which is not consistent with models of cloudlets moving isotropically within a spherical structure. Overall, only a smooth disk/slab-like model with systematic velocities remains consistent with the inferred properties of a single population of Lyα absorbers.

Kinetic analysis of 75selenium uptake by mitochondria of germinating Vigna radiata of different selenium status.

Earlier studies in our laboratory demonstrated the beneficial role of Se in Vigna radiata, a Se-deficient legume, during germination, as reflected in growth-related parameters and specific uptake of 75Se. Uptake of Na2(75)SeO3, added in vitro by mitochondria isolated from seedlings germinated in control (without Se), and Se-supplemented groups (0.5, 1.0, and 2.0 ppm Se) indicated a proportional increase in the uptake with added Na2(75)SeO3, in concentrations up to 25 microM. The uptake of 75Se, increased linearly with time up to 15 min and a definite efflux followed at 30 min. The results were indicative of cooperative effects during Se transport. Kinetic analyses of the uptake of 75Se during time intervals of 15 and 30 min were carried out both in the whole mitochondria and the mitochondrial protein fractions. Graphical analyses using Lineweaver-Burk plot, Hill plot, log [v] vs log [A] and Scatchard plot confirmed the existence of negative cooperativity during 75Se uptake. Hill coefficient (nH) values were estimated to be around 0.7-0.8. Scatchard plots for 75Se uptake were biphasic, suggesting the probable presence of two classes of binding sites. The number of high and low affinity binding sites were estimated to be around 4-7 and 26-30 nmol/mg protein, respectively. Studies with mitochondrial respiratory inhibitors indicated about 10-20% of the total 75Se uptake to be energy dependent. Inhibition of 75Se uptake by about 60-70% by sulfate and sulfite (5-25 microM) implies the involvement of dicarboxylate port in Se transport. A decrease in the uptake of 75Se by 40-60% effected by CdCl2, HgCl2, mersalyl, and NEM confirmed the interaction of thiols in the process. Evidence for the regulatory nature of 75Se uptake by mitochondria of V. radiata emerges from the present study.

Veratrine-induced tetanic contracture of the rat isolated left atrium

An investigation has been made of the putative direct myocardial protective effects of the alpha 1-adrenoceptor antagonists, prazosin and WB4101, against tetanic contractures of rat isolated left atria following modified Na+ channel function and consequent Ca2+ loading elicited by veratrine. Veratrine evoked concentration-dependent, reversible, tetanic contractures which were critically dependent upon the external Ca2+ concentration. Tetrodotoxin (TTX), prazosin, WB 4101 and R 56865 (0.1-10 microM) prevented tetanic contracture elicited by veratrine (100 micrograms/ml) at concentrations which were significantly lower than those which decreased active tension development. The apparent Hill coefficients (nH) obtained for TTX, prazosin, WB 4101 and R 56865 were comparable (range 0.79-0.93), and are consistent with a single site of action. In contrast, the class 1 antiarrhythmic agents, quinidine and lidocaine, elicited no significant inhibition of veratrine-induced contracture at 30 microM, but almost completely abolished the contractures at 100 microM. The nH values for quinidine and lidocaine were found to be significantly greater than unity (3.1 and 2.6, respectively). The L-type Ca2+ channel blockers, diltiazem, nicardipine, nifedipine and verapamil only weakly prevented tetanic contracture, whilst markedly, and concentration-dependently, decreasing active tension development. Neither atropine (10 microM) nor propranolol (1 microM) significantly modified either veratrine-induced contractures or active tension development. In conclusion, evidence is presented of novel, direct protective effects of prazosin and WB 4101 against tetanic contracture following modified Na+ channel function and Ca2+ loading provoked by veratrine. The precise mechanisms involved are unclear at present, but appear to be distinct from blockade of atrial alpha 1-adrenoceptors or L-type Ca2+ channels.(ABSTRACT TRUNCATED AT 250 WORDS)

Computer modelling of estrogenic transcriptional activation can account for different types of dose-response curves of estrogens

Estrogenic activity of diphenylethanes and -ethenes was determined by uterine growth in immature mice and analyzed by weighed regression of logit-transformed effect on log dose values. This resulted in a range of Hill coefficients nh from 0.3 to 2 corresponding to the molecular mechanism of estrogenic transcriptional activation. Binding of agonists (hormones, H) to estrogen receptors (ER) lead to receptor dimerization depending on the structure of the ligand. Three hormone-receptor complexes, H-ER, H-ER-ER, and H-ER-ER-H, which bind with different affinity to short palindromic DNA sequences (estrogen responsive elements), can be proposed. Transcriptional activating functions of the DNA-bound ER are subsequently induced. We have derived an equilibrium model including these steps. Computer simulations of Hill plots based on the model have completely reproduced the range of observed nh values. Hill coefficients are > 1.5 if the homodimer H-ER-ER-H and < 0.7 if the heterodimer H-ER-ER strongly predominates. If ER dimerization is disturbed (H-ER monomer predominant), nH is closer to 1. Hill coefficients and pD2 values (negative decadic logarithms of molar estrogen doses causing 50% of the maximal effects) are related to parameters of ER dimerization and the two steps of hormone-receptor dissociation. When a series of 1,2-bis(3′-or 4′-hydroxyphenyl)ethanes and -ethenes is studied, a rather simple dependence of nH and pD2 on the nature of alkyl groups symmetrically substituted at C-atoms 1 and 2 can be observed. In terms of the model this implies that ethyl and α-branched higher alkyl substituents (nH ⪢ 1) appear to stabilize the homodimer, while methyl and CF3 groups (nH ⪡ 1) could lead to a rapid dissociation of the homodimer to the heterodimer. With longer n-alkyl and β-branched alkyl substitution (nHfrom 0.66 to 1.3), dimerization itself can be limited or the ligand-homodimer dissociation is only moderately increased. Thus, a strong sterical constraint could exist with respect to the stabilization of the second ligand-receptor bond in the homodimer.

1H- and 13C-NMR studies of solutions of hyaluronic acid esters and salts in methyl sulfoxide: comparison of hydrogen-bond patterns and conformational behaviour

The 1H- and 13C-NMR spectra of the ethyl and benzyl esters and the tetrabutylammonium and tetraethylammonium salts of hyaluronic acid { ▪ 2)-β- d-Glc pA-(1 → 3)-β- d-Glc pNAc-(1 ▪ n } in Me 2SO- d 6 have been assigned using 1D and 2D techniques. The chemical shifts of the resonance of GlcNAc C-3 suggest that the relative orientations of the monosaccharides at the (1 → 3) linkage in the esters and salts are different. Small differences in the chemical shifts of the resonance GlcA C-4 suggest only a slight conformational variation around the (1 → 4) linkage. The 13C-NMR data also suggest similarities in conformation between the esters in Me 2SO- d 6 and the salts in water. The chemical shifts of the 1H resonances for NH and OH groups and their temperature dependence for the esters and salts in Me 2SO reveal markedly stronger inter-residue hydrogen bonds between the carboxyl and NH groups and between HO-4 of GlcA and O-5 of GlcNAc for the salts. The 3 J 2,NH values indicate a slightly different orientation for the acetamido group. For solutions in Me 2SO, the higher segmental flexibility of the esters is supported by the line widths, whereas the reduced viscosity for the tetrabutylammonium salt showed a sigmoidal concentration dependence and suggests association of chains which could contribute to the segmental rigidity. The linear concentration dependence for the benzyl ester suggests a higher overall flexibility without chain association.

Structural Properties of High Electronic Quality a-Si1-xCx:H by Infrared Spectroscopy

ABSTRACTInfrared (ir) spectroscopy is used to investigate the structural properties of a-SiC:H in a wide compositional range and as a function of film thickness. Hydrogen content NH increases considerably with increasing carbon fraction. For low carbon alloys this is mainly due to an increase of hydrogen bonded to silicon, incorporated in a mono- or dihydride form. Above Eg=2.3eV the proportion of hydrogen incorporated in C-H bonds increases considerably. Oxidation of high C alloys is observed. Converting experimental transmission exactly into absorption data yields thickness independent NH values. It is shown that the previously reported discrepancy between the hydrogen content calculated from ir and nuclear reaction techniques is an artifact of the ir analysis.

Variations in the kinetic response of several different phosphate-dependent glutaminase isozymes during acute metabolic acidosis

We describe the kinetic modifications to mitochondrial-membrane-bound phosphate-dependent glutaminase in various types of rat tissue brought about by acute metabolic acidosis. The activity response of phosphate-dependent glutaminase to glutamine was sigmoidal, showing positive co-operativity, the Hill coefficients always being higher than 2. The enzyme from acidotic rats showed increased activity at subsaturating concentrations of glutamine in kidney tubules, as might be expected, but not in brain, intestine or liver tissues. Nevertheless, when brain and intestine from control rats were incubated in plasma from acutely acidotic rats enzyme activity increased at 1 mM glutamine in the same way as in kidney cortex. The enzyme from liver tissue remained unaltered. S0.5 and nH values decreased significantly in kidney tubules, enterocytes and brain slices preincubated in plasma from acidotic rats. The sigmoidal curves of phosphate-dependent glutaminase shifted to the left without any significant changes in Vmax. The similar response of phosphate-dependent glutaminase to acute acidosis in the kidney, brain and intestine confirms the fact that enzymes from these tissues are kinetically identical and reaffirms the presence of an ammoniagenic factor in plasma, either produced or concentrated in the kidneys of rats with acute acidosis.

Sulfhydryl Groups Modulate the Allosteric Interaction Between Glycine Binding Sites at the Inhibitory Glycine Receptor

We have investigated the effect of chemical reagents that modify sulfhydryl groups on the ligand binding properties of the glycine receptor (GlyR). The Hill coefficient (nH) for the displacement of [3H]strychnine binding by glycine was increased from approximately 0.8 to values significantly above 1 (approximately 1.2-1.4) in membranes pretreated with the disulfide-reducing agent dithiothreitol or glutathione. However, the affinity of strychnine or glycine for the GlyR was not affected by these treatments. This indicates that several glycine binding sites interact cooperatively for displacing bound strychnine under such experimental circumstances. A similar increase in the nH for glycine has been observed when the temperature of the binding assay was increased to 37 degrees C. Combination of dithiothreitol pretreatment and increased binding temperature led to nH variations similar to those observed with either of these treatments alone, a finding suggesting that their mechanisms of action are not independent. Conversely, modification of rat spinal cord membranes or of purified and reconstituted GlyR preparations with the sulfhydryl-alkylating agent N-ethylmaleimide or fluorescein-maleimide decreased nH values to approximately 0.5, without affecting glycine or strychnine affinities. This effect may be caused by an increased heterogeneity of GlyR populations. It is interesting that occupancy of the receptor by glycine or beta-alanine (but not by antagonists) specifically protects from the effects of the different sulfhydryl reagents. Moreover, the presence of some of the Eccles' anions, i.e., anions that permeate through the channels associated with GlyRs and gamma-aminobutyric acidA receptors, seems to be required for the action of both dithiothreitol and N-ethylmaleimide.(ABSTRACT TRUNCATED AT 250 WORDS)

Site-specific Mutation of Tyr240 → Phe in the Catalytic Chain of Escherichia coli Aspartate Transcarbamylase: Consequences for kinetic mechanism

Abstract In the catalytic chain of Escherichia coli aspartate transcarbamylase, Tyr240 helps stabilize the T-state conformation by an intrachain hydrogen bond to Asp271. Changes in kinetic characteristics of ATCase that result from disruption of this bond by site-specific mutation of Tyr240----Phe have been investigated by isotopic exchanges at chemical equilibrium. The Tyr240----Phe (Y240F) mutation caused the rate of the [32P] carbamyl phosphate (C-P) in equilibrium Pi exchange to decrease by 2-8-fold, without altering the [14C]Asp in equilibrium N-carbamyl-L-aspartate (C-Asp) rate. The mutation also caused the S0.5 and Hill nH values to decrease in virtually every substrate saturation experiment. Upon increasing the concentrations of the C-P,Pi or C-P,C-Asp reactant-product pairs, inhibition effects observed with the C-P in equilibrium Pi exchange for wild-type enzyme were not apparent with the Y240F mutant enzyme. In contrast, upon increasing the concentrations of the Asp,C-Asp and Asp,Pi pairs, inhibition effects on C-P in equilibrium Pi observed with wild-type enzyme became stronger with the Y240F mutant enzyme. These data indicate that the Tyr240----Phe mutation alters the kinetic mechanism in two different ways: on the reactant side, C-P binding prior to Asp shifts from preferred to compulsory order, and, on the product side, C-Asp and Pi release changes from preferred to nearly random order. These conclusions were also confirmed on a quantitative basis by computer simulations and fitting of the data, which also produced an optimal set of rate constants for the Y240F enzyme. The Arrhenius plot for wild-type holoenzyme was biphasic, but those for catalytic subunits and Y240F enzyme were linear (monophasic). Taken together, the data indicate that the Tyr240----Phe mutation destabilizes the T-state and shifts the equilibrium for the T-R allosteric transition toward the R-state by increasing the rate of T----R conversion.

Sulfate-activating Enzymes of Penicillium chrysogenum: The ATP Sulfurylase Adenosine 5′-Phosphosulfate Complex does not Serve as a Substrate for Adenosine 5′-Phosphosulfate Kinase

Abstract At a noninhibitory steady state concentration of adenosine 5'-phosphosulfate (APS), increasing the concentration of Penicillium chrysogenum ATP sulfurylase drives the rate of the APS kinase-catalyzed reaction toward zero. The result indicates that the ATP sulfurylase.APS complex does not serve as a for APS kinase, i.e. there is no substrate channeling of APS between the two sulfate-activating enzymes. APS kinase had no effect on the [S]0.5 values, nH values, or maximum isotope trapping in the single turnover of ATP sulfurylase-bound [35S]APS. Equimolar APS kinase (+/- MgATP or APS) also had no effect on the rate constants for the inactivation of ATP sulfurylase by phenylglyoxal, diethylpyrocarbonate, or N-ethylmaleimide. Similarly, ATP sulfurylase (+/- ligands) had no effect on the inactivation of equimolar APS kinase by trinitrobenzene sulfonate, diethylpyrocarbonate, or heat. (The last promotes the dissociation of dimeric APS kinase to inactive monomers.) ATP sulfurylase also had no effect on the reassociation of APS kinase subunits at low temperature. The cumulative results suggest that the two sulfate activating enzymes do not associate to form a 3'-phosphoadenosine 5'-phosphosulfate synthetase complex.