A thermostable bacterial cocaine esterase rapidly eliminates cocaine from brain in nonhuman primates.

A long-acting, thermostable bacterial cocaine esterase (CocE) has been identified that rapidly degrades cocaine with a K(M) of 1.33+0.085 µM. In vivo evaluation of CocE has shown protection against convulsant and lethal effects of cocaine in rodents, confirming the therapeutic potential of CocE against cocaine overdose. However, the current study is the first to evaluate the effects of CocE on cocaine brain levels. Positron emission tomogrpahy neuroimaging of [(11)C]cocaine was used to evaluate the time course of cocaine elimination from brain in the presence and absence of CocE in nonhuman primates. Systemic administration of CocE eliminated cocaine from the rhesus-monkey brain approximately three times faster than control conditions via peripheral actions through attenuating the input function from blood plasma. The efficiency of this process is sufficient to alleviate or prevent adverse central nervous system effects induced by cocaine. Although the present study used tracer doses of cocaine to access brain clearance, these findings further support the development of CocE for the treatment of acute cocaine toxicity.

Oestradiol alters central 5-HT1A receptor binding potential differences related to psychosocial stress but not differences related to 5-HTTLPR genotype in female rhesus monkeys.

Social subordination in female macaques represents a well-described model of chronic psychosocial stress. Additionally, a length polymorphism (5-HTTLPR) in the regulatory region of the serotonin (5-HT) transporter (5-HTT) gene (SLC6A4) is present in rhesus macaques, which has been linked to adverse outcomes similar to that described in humans with an analogous 5-HTTLPR polymorphism. The present study determined the effects of social status and the 5-HTTLPR genotype on 5-HT1A receptor binding potential (5-HT1A BP(ND)) in brain regions implicated in emotional regulation and stress reactivity in ovariectomised female monkeys, and then assessed how these effects were altered by 17ß-oestradiol (E(2)) treatment. Areas analysed included the prefrontal cortex [anterior cingulate (ACC); medial prefrontal cortex (mPFC); dorsolateral prefrontal cortex; orbitofrontal prefrontal cortex], amygdala, hippocampus, hypothalamus and raphe nucleui. Positron emission tomography using p-[(18) F]MPPF was performed to determine the levels of 5-HT1A BP(ND) under a non-E(2) and a 3-week E(2) treatment condition. The short variant (s-variant) 5-HTTLPR genotype produced a significant reduction in 5-HT1A BP(ND) in the mPFC regardless of social status, and subordinate s-variant females showed a reduction in 5-HT1A BP(ND) within the ACC. Both these effects of 5-HTTLPR were unaffected by E(2). Additionally, E(2) reduced 5-HT1A BP(ND) in the dorsal raphe of all females irrespective of psychosocial stress or 5-HTTLPR genotype. Hippocampal 5-HT1A BP(ND) was attenuated in subordinate females regardless of 5-HTTLPR genotype during the non-E(2) condition, an effect that was normalised with E(2). Similarly, 5-HT1A BP(ND) in the hypothalamus was significantly lower in subordinate females regardless of 5-HTTLPR genotype, an effect reversed with E(2). Taken together, the data indicate that the effect of E(2) on modulation of central 5HT1A BP(ND) may only occur in brain regions that show no 5-HTTLPR genotype-linked control of 5-HT1A binding.

CRH receptor antagonism reverses the effect of social subordination upon central GABAA receptor binding in estradiol-treated ovariectomized female rhesus monkeys.

Persistent exposure to environmental stressors causes dysregulation of the limbic-hypothalamic-pituitary-adrenal (LHPA) axis and alters GABAA receptor (GABAAR) levels throughout the brain. Social subordination in socially housed female rhesus results in distinctive stress-related physiological and behavioral phenotypes that are dependent on the ovarian hormone estradiol (E2). In the present study, we utilized ovariectomized adult female rhesus monkeys undergoing hormone replacement with E2 to test the hypothesis that the chronic psychosocial stress of subordination alters GABAAR binding potential (GABAAR BPND) in limbic regions implicated in emotional processing including the prefrontal cortex, temporal lobe (amygdala and hippocampus), and hypothalamus. Furthermore, we tested the hypothesis that peripheral administration of a corticotropin-releasing hormone (CRH) receptor antagonist (astressin B) would reverse the alterations in GABAAR binding within these regions in subordinate females. After subjects received astressin B or saline for three consecutive days, GABAAR BPND was determined by positron emission tomography (PET) using (18)F-flumazenil as a radioligand. T1-weighted structural magnetic resonance imaging scans were also acquired for PET scan co-registration, in order to perform a region of interest analysis using the pons as a reference region. Compared to socially dominant females, subordinate females exhibited increased GABAAR BPND in the prefrontal cortex but not in the temporal lobe or the hypothalamus. Administration of astressin B eliminated the status difference in GABAAR BPND in the prefrontal cortex, suggesting that the chronic stressor of social subordination modulates GABAergic tone via effects on CRH and the LHPA axis, at least in prefrontal regions.

The relation of developmental changes in brain serotonin transporter (5HTT) and 5HT1A receptor binding to emotional behavior in female rhesus monkeys: effects of social status and 5HTT genotype.

The goal of the present study was to examine how social subordination stress and 5HTT polymorphisms affect the development of brain serotonin (5HT) systems during the pubertal transition in female rhesus monkeys. We also examined associations with developmental changes in emotional reactivity in response to a standardized behavioral test, the Human Intruder (HI). Our findings provide the first longitudinal evidence of developmental increases in 5HT1A receptor and 5HTT binding in the brain of female primates from pre- to peripuberty. The increase in 5HT1A BP(ND) in these socially housed female rhesus monkeys is a robust finding, occurring across all groups, regardless of social status or 5HTT genotype, and occurring in the left and right hemispheres of all prefrontal regions studied, as well as the amygdala, hippocampus, hypothalamus, and raphe nuclei. 5HTT BP(ND) also showed an increase with age in raphe, anterior cingulate cortex, and dorsolateral prefrontal cortex. These changes in brain 5HT systems take place as females establish more adult-like patterns of social behavior, as well as during the HI paradigm. Indeed, the main developmental changes in behavior during the HI (increase in freezing and decrease in submission/appeasement) were related to neurodevelopmental increases in 5HT1A receptors and 5HTT, because the associations between these behaviors and 5HT endpoints emerge at peripuberty. We detected an effect of social status on 5HT1A BP(ND) in the hypothalamus and on 5HTT BP(ND) in the orbitofrontal cortex, with subordinates showing higher BP(ND) than dominants in both cases during the pubertal transition. No main effects of 5HTT genotype were observed for 5HT1A or 5HTT BP(ND). Our findings indicate that adolescence in female rhesus monkeys is a period of central 5HT reorganization, partly influenced by exposure to the social stress of subordination, that likely functions to integrate adrenal and gonadal systems and shape the behavioral response to emotionally challenging social situations.

2,5-O-methylene-D-mannitol: two polymorphs and the NaI complex.

Two polymorphs of the title compound, (4R,5R,6R,7R)-4,7-bis(hydroxymethyl)-1,3-dioxepane-5,6-diol, C(7)H(14)O(6), both have Z' = 2 at 100 K, and differ in their hydrogen-bonding patterns. The sodium iodide complex, NaI.C(7)H(14)O(6), is isomorphous with the NaCl complex, and has the mannitol, cation and anion all lying on twofold axes. The dioxepane rings of all three molecules are in the twist-chair conformation.

2,3,4,5-Tetra-O-acetyl-1,6-di-O-(triphenylmethyl)-D-mannitol.

The central six-C-atom chain of the title compound, C52H50O10, adopts a nearly planar extended conformation free from C parallel C, C parallel O and O parallel O 1,3-parallel interactions. The three torsion angles formed by these atoms have values of 177.4 (2), 174.2 (2) and -178.0 (2) degrees. The bulky triphenylmethyl groups, which are oriented gauche to their neighboring acetoxy groups with O--C--C--O torsion angles of -69.8 (2) and -63.0 (2) degrees, cause no distortion of the bond lengths and bond angles of the sugar moiety.

3,4-di-O-acetyl-2,5-anhydro-1,6-di-O-(p-tolylsulfonyl)-D-mannitol.

The title compound, 2,5-bis(4-methylphenylsulfonyloxy-methyl)oxolane-3,4-diyl diacetate, C24H28O11S2, lies on a crystallographic twofold axis. It adopts a perfect twist 3(4)T conformation in the solid state. The puckering parameters of the tetrahydrofuran ring are q = 0.32 (8) A and rho = 92.3 (5) degrees. The acetyl groups are planar, with the non-H atoms deviating less than 0.002 (3) A from their mean plane. They have an (S)-cis conformation with the C-O and C = O bonds eclipsed, and each acetyl group is orientated with the C = O group syndiaxial to the C-H bond at the ring C atom to which the group is attached.

Crystal structure of 2,5-anhydro-1-O-(p-tolylsulfonyl)-D-mannitol.

2,5-Anhydro-1-O-(p-tolylsulfonyl)-D-mannitol, C13H18SO7, Mr = 318.4, monoclinic, C2, a = 26.370(6), b = 7.9741(11), c = 6.6801(6) A, beta = 91.401(11) degrees, V = 1404.3(6) A3, Z = 4, DX = 1.506 g/cm3, CuK alpha, lambda = 1.54184 A, mu = 23.03 cm-1, F(000) = 672, T = 296(1) K, R = 0.042 for 2832 observations with I > 3 sigma (I) (of 2864 unique data). On the esterified side of the molecule, three bond lengths and three bond angles show small changes compared to the unesterified side, which is similar to the symmetrical parent compound, 2,5-anhydro-D-mannitol. The conformation of the five-membered ring is E5 with P = 49.3 degrees and tau m = 38.1 degrees. The hydroxymethyl groups adopt g+ and g- dispositions similar to the parent molecule. The three hydroxyl groups are involved in a network of intermolecular hydrogen bounds both as donors and acceptors.

Structure of 2,5:3,4-dianhydro-D-altritol.

3,6-Dioxabicyclo[3.1.0]hexane-2,4-dimethanol, C6H10O4, M(r) = 146.1, orthorhombic, P2(1)2(1)2(1), a = 7.6209 (2), b = 9.1292 (3), c = 9.6135 (5) A, V = 668.8 (1) A3, Z = 4, Dx = 1.451 g cm-3, lambda(Mo K alpha) = 0.71073 A, mu = 1.15 cm-1, F(000) = 312, T = 298 K, R = 0.029 for 1280 observations with I greater than 3 sigma(I) (of 1695 unique data). The tetrahydrofuran ring has the envelope conformation, OE, with P of 94.3 degrees and tau m = 24.0 degrees. C atoms deviate from their best plane by +/- 0.0006 (1) to 0.010 (1) A, and the O atom lies 0.331 (1) A from that plane. The epoxide O atom is syn to the tetrahydrofuran O atom. Each hydroxy group is involved in intermolecular hydrogen bonding both as donor and acceptor. The two hydrogen bonds have O...O distances of 2.743 (1) and 2.729 (1) A, and angles about H of 166.3 (12) and 172 (2) degrees, respectively.

Synthesis and X-ray crystal and solution structures of 2,5-anhydro-3,4-O-(1,2-ethanediyl)-D-mannitol: a locked 4T3 furanose conformer.

2,5-Anhydro-3,4-O-(1,2-ethanediyl)-D-mannitol (1) was prepared from 2,5-anhydro-D-mannitol (2) in three steps. The fused ring system was introduced by a phase-transfer alkylation using 1,2-dibromoethane. Its conformation in solution was determined by NMR studies at 500 MHz. Variable-temperature studies showed no lineshape change from 25 to 80 degrees in D2O. The data indicate that the five-membered ring is locked by the trans-fused six-membered 1,4-dioxane ring into a twist 4T3 conformation. A single-crystal X-ray study was carried out. The crystals are orthorhombic, C222(1), a = 4.7252 (6), b = 14.0364 (12), c = 13.268 (2) A, Z = 4, with R = 0.032 for 894 observations. The molecule lies upon a crystallographic two-fold axis, and thus the five-membered ring exists in a perfect 4T3 conformation with a pseudorotation angle of 0 degree and amplitude of 47.2 degrees, in agreement with the NMR results. We have shown earlier that, among twenty possible conformers, phosphofructokinase acts specifically on the 4T3 conformer of the beta anomer of D-fructose 6-phosphate.

Structure of 1,2,3,4,5,6-hexa-O-acetyl-myo-inositol.

C18H24O12, Mr = 432.4, monoclinic, Cc, a = 8.996 (3), b = 20.890 (6), c = 11.872 (4) A, beta = 101.11 (2) degrees, V = 2189 (1) A3, Z = 4, Dx = 1.312 g cm-3, Mo K alpha, lambda = 0.71069 A, mu = 1.05 cm-1, F(000) = 912, T = 163 K, R = 0.041, wR = 0.0375 for 2158 reflections (Fo greater than or equal to 6 sigma magnitude of Fo). The ring is in the chair conformation 4C1 with five equatorial groups and one axial group bonded to C(2) as expected. The carbonyl bonds of the acetate groups at positions 2, 4, 5 and 6 are approximately coplanar with their respective ring C-H bonds. However, those at positions 1 and 3 are rotated towards the H(2) atom.

Two-dimensional 1H-, 13C-, and 31P-nuclear magnetic resonance and molecular-mechanics investigation of D-fructose 2,6-bisphosphate.

Two-dimensional nuclear magnetic resonance studies have been carried out to assign unequivocally all the proton, carbon, and phosphorus resonances of D-fructofuranose 2,6-bisphosphate (1) and to verify its structure using a 400-MHz spectrometer. Several unexpected chemical-shift values and coupling constants were obtained. Molecular mechanics calculations (Sybyl) carried out to minimize the conformational energy of 1 yield phi C-1,P-2 = + 84, phi C-3,P-2 = - 155, and phi C-5,P-6 = + 175. Thus the unusual near-gauche orientations of C-1 and C-3 to P-2 in 1 can explain their small vicinal coupling constants (3JC-1,P-2 = 1.2, and 3JC-3,P-2 = 3.8 Hz), in contrast to the expected larger value seen for 3JC-5,P-6 namely, 6.9 Hz. Treatment of a sample of this compound with sodium borohydride did not affect its nuclear magnetic resonance spectrum, substantiating that O-2 is phosphorylated. Oxidation with sodium periodate yielded an intermediate which decomposed by a beta-elimination mechanism involving the 6-phosphate group. These data establish unequivocally the 1H, 13C, and 31P assignments and explain the observed anomalous shifts. Moreover they indicate that the activator of fructose 6-phosphate 1-kinase is the beta anomer of the 4T3 conformer of D-fructose 2,6-bisphosphate.

The X-ray crystal structure of 2,3:4,5-di-O-isopropylidene-1-O-methyl-beta-D-fructopyranose.

2,3:4,5-Di-O-isopropylidene-1-O-methyl-beta-D-fructopyranose, C13H22O6, Mr = 274.3, orthorhombic, P212121, a = 12.388 (2), b = 13.307 (5), c = 8.660 (1) A, V = 1427.4 (9) A3, Z = 4, Dm = 1.24 g.cm-3, Dx = 1.276 g.cm-3, CuKalpha, lambda = 1.54184 A, mu = 8.0 cm-1, F(000) = 592, T = 295 (1) K, R = 0.032 for 1586 observations (of 1693 unique data). The molecule is a derivative of the naturally occurring carbohydrate D-fructose. The data reported here indicate that the ketose six-membered ring is constrained by the presence of two fused five-membered rings into the 3SO conformation. These findings agree with the n.m.r.-spectroscopic results for 2,3:4,5-di-O-benzylidene-beta-D-fructopyranose. As a result of crystal packing forces, the exocyclic side-chain has a C-C-O-C torsion angle of -102 degrees, quite different from the expected value of 180 degrees.

The X-ray crystal structure of DL-(1,3,5/2,4)-1,2,3,4-tetraacetoxy-5-(acetoxymethyl)cyclohexane.

The crystal and molecular structure of the pseudo-sugar DL-(1,3,5/2,4)-1,2,3,4-tetraacetoxy-5-(acetoxymethyl)cyclohexane ("pseudo-beta-DL-glucopyranose pentaacetate") has been determined by X-ray diffraction and statistical-phasing procedures. The data were refined to R = 0.049 and Rw = 0.054 over 1543 reflections with I greater than 3 sigma(I). The racemic crystals are monoclinic, space group P2(1)/c, a = 11.580(2), b = 8.276(1), c = 22.031(2) A, beta = 104.33(1) degrees, Dc = 1.26 g/cm3, with four molecules in the unit cell. The ring has a 4C1(D), 1C4(L) conformation, with puckering amplitude of 0.582(4) A, a pseudorotation angle of -168.35 degrees, and a gg orientation about the C-5-C-6 bond.

Carbohydrate substrate specificity of bacterial and plant pyrophosphate-dependent phosphofructokinases.

Pyrophosphate-dependent phosphofructokinase from the facultative anaerobic bacterium Propionibacterium freudenreichii and from the mung bean Phaseolus aureus has been purified to homogeneity. Potential utilization of carbohydrate substrate analogues for each enzyme was initially screened by using Fourier transform 31P NMR at pH 8 and 25 degrees C and monitoring the appearance of the phosphate resonance in the direction of D-fructose 6-phosphate phosphorylation (forward reaction direction) and, with the bisphosphate analogues, the appearance of the pyrophosphate resonance in the direction of phosphate phosphorylation (reverse reaction direction). Both enzymes are strict in their requirements for the sugar phosphate substrate, with only D-fructose 6-phosphate, D-sedoheptulose 7-phosphate, and 2,5-anhydro-D-mannitol 6-phosphate, or their respective bisphosphates in the reverse reaction direction, utilized as substrates at detectable levels. The dissociation constants for D-psicose 6-phosphate, D-tagatose 6-phosphate, and L-sorbose 6-phosphate are an order of magnitude larger than that for D-fructose 6-phosphate, indicating a stringent steric requirement for the D-threo (trans) configuration at the two nonanomeric furan ring hydroxyl groups. These results strongly suggest that the anomeric, epimeric, and tautomeric form of the sugar phosphate substrates favored by both enzymes is the beta-D-fructofuranose form. Dissociation constants for nonsubstrate analogues were used to provide information on the nature of the active site. Competitive inhibition patterns vs. fructose 1,6-bisphosphate were obtained for a series of 1,n-alkanediol bisphosphates (where n = 2-9).(ABSTRACT TRUNCATED AT 250 WORDS)

Kinetic studies on the activation of pyrophosphate-dependent phosphofructokinase from mung bean by fructose 2,6-bisphosphate and related compounds.

Pyrophosphate-dependent phosphofructokinase (PPi-PFK) was purified from the mung bean Phaseolus aureus. The enzyme is activated by fructose 2,6-bisphosphate at nanomolar concentrations. The enzyme exhibits Michaelis-Menten kinetics, and the reaction mechanism, deduced from initial velocity studies in the absence of inhibitors as well as product and dead-end inhibition studies, is rapid equilibrium random in the presence and absence of fructose 2,6-bisphosphate. In the direction of fructose 6-phosphate phosphorylation, saturating fructose 2,6-bisphosphate (1 microM) increases V congruent to 9-fold and increases V/KMgPPi and V/KF6P about 30-fold. In the reverse direction (phosphate phosphorylation), the same concentration of activator has little if any effect on V or the Km for inorganic phosphate (Pi) and Mg2+ but does increase V/KFBP about 42-fold. No changes were observed in any of the other rate constants. The binding affinity of fructose 2,6-bisphosphate to all enzyme forms is identical. The activator site of the mung bean PPi-PFK binds fructose 2,6-bisphosphate with a Kact of 30 nM with the 2,5-anhydro-D-glucitol 1,6-bisphosphate (the most effective analogue) 33-fold less tightly. Of the alkanediol bisphosphate series, 1,4-butanediol bisphosphate exhibited the tightest binding (Kact congruent to 3 microM). These and a series of other activating analogues are discussed in relation to the activator site.

Stereospecificity of the fructose 2,6-bisphosphate site of muscle 6-phosphofructo-1-kinase.

We explored the stereospecificity of the fructose 2,6-bisphosphate site of rabbit muscle 6-phosphofructo-1-kinase by determination of the activation constants (Ka) of several structurally locked analogues of this potent metabolic regulator. Under the assay conditions used, the Ka of fructose 2,6-bisphosphate was 0.12 microM. The most effective synthetic analogues and their Ka's were 2,5-anhydro-D-mannitol 1,6-bisphosphate (2.9 microM), 1,4-butanediol bisphosphate (6.6 microM), hexitol 1,6-bisphosphate (40 microM), and 2,5-anhydro-D-glucitol 1,6-bisphosphate (47 microM). Ten other bisphosphate compounds were much less effective as activators of the enzyme. These findings indicate that, unlike its active site, this allosteric site of 6-phosphofructo-1-kinase does not require the furanose ring. Its basic requirement seems to be a compound with two phosphate groups approximately 9 A apart. Although the free hydroxy groups of the activator do not seem to be essential, their presence enhances appreciably the affinity of the ligand for this regulatory site.