Molecular cocrystals of carboxylic acids. XXVII - An investigation into the use of triphenylphosphine oxide cocrystals for non-linear optics: the crystal structure of the adduct of triphenylphosphine oxide with N-methylpyrrole-2-carboxylic acid

Four adducts of triphenylphosphine oxide with aromatic carboxylic acids have been synthesized and tested for second-order non-linear optical properties. These were with N-methylpyrrole-2-carboxylic acid (I), indole-2-carboxylic acid (2), 3-dimethylaminobenzoic acid (3), and thiophen-2-carboxylic acid (4). Compound (1) produced clear, colourless crystals (space group P2(1)2(1)2(1) With a 9.892(1), b 14.033(1), c 15.305(1) Angstrom, Z 4) which allowed the structure to be determined by X-ray diffraction.

Nucleotide sequence of the nifH gene coding for nitrogen reductase in the acetic acid bacterium Acetobacter diazotrophicus

The nifH gene sequence of the nitrogen-fixing bacterium Acetobacter diazotrophicus was determined with the use of the polymerase chain reaction and universal degenerate oligonucleotide primers. The gene shows highest pair-wise similarity to the nifH gene of Azospirillum brasilense. The phylogenetic relationships of the nifH gene sequences were compared with those inferred from 16S rRNA gene sequences. Knowledge of the sequence of the nifH gene contributes to the growing database of nifH gene sequences, and will allow the detection of Acet. diazotrophicus from environmental samples with nifH gene-based primers.

cis-3-hydroxy-N-methyl-L-proline: a new amino acid from a southern Australian marine sponge, Dendrilla sp.

A specimen of the sponge Dendrilla sp. collected during commercial trawling operations in the Great Australian Eight, Australia, analyses for a very high natural abundance of the new amino acid cis-3-hydroxy-N-methyl-L-proline (1). The complete stereostructure for (1) was determined by spectroscopic analysis and chemical derivatization.

New stereoselective routes to macrocyclic ligands

Reaction of bis(ethane-1,2-diamine)copper(II) with acetaldehyde and nitromethane in methanol leads, stereoselectively, to the new macrocyclic complex (trans-5(R),7(R),12(S),14(S))-tetramethyl-6,13-dinitro-1,4,8,11-tetraazacyclotetradecane)copper(II) perchlorate alpha-[CuL1](ClO4)(2) in good yield. Reduction of the nitro groups affords the hexaamine (L-2), which was crystallized as [H4L2](ClO4)(4) . 2H(2)O and characterized by an X-ray crystal structure study (monoclinic P2(1)/n, a = 9.763(2) Angstrom, b = 12.1988(7) Angstrom, c = 13.036(2) Angstrom, beta = 105.668(7)degrees, Z = 2) and complexed with Cu-II to produce the complex beta-[Cu(H2L2)](ClO4)(4) . 2H(2)O, which has also been characterized by X-ray crystallography (monoclinic P2(1)/n, a = 9.717(4) Angstrom, b = 12.174(2) Angstrom, c = 13.036(5) Angstrom, beta = 106.51(2)degrees, Z = 2). Reaction of alpha-[CuL1](2+) with either basic hydrogen peroxide or dilute nitrous acid leads to mild reduction of the nitro groups to afford the ketoxime L-3 as its N-based isomeric Cu-II complexes, trans-I [CuL3](ClO4)(2) and trans-II [Cu(L-3)Cl]Cl . 7H(2)O, the latter of which has been characterized structurally: triclinic, <P(1)over bar> a = 10.8441(5) Angstrom, b = 11.6632(9) Angstrom, c = 11.8723(9) Angstrom, alpha = 113.634(7)degrees, beta = 95.744(5), gamma = 94.851(5)degrees Z = 2. Variations in the configurations of the coordinated amines in [CuL1](2+), [CuL2](2+), and [CuL3](2+) have a profound effect on the spectroscopy and electrochemistry of their complexes.

Stage-specific proteophosphoglycan from Leishmania mexicana amastigotes - Structural characterization of novel mono-, di-, and triphosphorylated phosphodiester-linked oligosaccharides

Intracellular amastigotes of the protozoan parasite Leishmania mexicana secrete a macromolecular proteophosphoglycan (aPPG) into the phagolysosome of their host cell, the mammalian macrophage. The structures of aPPG glycans were analyzed by a combination of high pH anion exchange high pressure liquid chromatography, gas chromatography-mass spectrometry, enzymatic digestions, electrospray-mass spectrometry as well as H-1 and P-31 NMR spectroscopy. Some glycans are identical to oligosaccharides known from Leishmania mexicana promastigote lipophosphoglycan and secreted acid phosphatase, However, the majority of the aPPG glycans represent amastigote stage-specific and novel structures. These include neutral glycans ([Glc beta(1-3)](1-2)Gal beta 1-4Man, Gal beta 1-3Gal beta 1-4Man, Gal beta 1-3Glc beta 1-3Gal beta 1-4Man), several monophosphorylated glycans containing the conserved phosphodisaccharide backbone (R-3-[PO4-6-Gal]beta 1-4Man) but carrying stage-specific modifications (R = Gal beta 1-, [Glc beta 1-3](1-2)Glc beta 1-), and monophosphorylated aPPG tri- and tetrasaccharides that are uniquely phosphorylated on the terminal hexose (PO4-6-Glc beta 1-3Gal beta 1-4Man, PO4-6-Glc beta 1-3Glc beta 1-3Gal beta 1-4Man, PO4-6-Gal beta 1-3Glc beta 1-3Gal beta 1-4Man), In addition aPPG contains highly unusual di- and triphosphorylated glycans whose major species are PO4-6-Glc beta 1-3Glc beta 1-3[PO4-6-Gal]beta 1-4Man, PO4-6-Gal beta 1-3Glc beta 1-3 [PO4-6-Gal]beta 1-4Man, PO4-6-GaL beta 1-3Glc beta 1-3Glc beta 1-3[PO4-6-Gal]beta 1-4Man, PO4-6-Glc beta 1-3[PO4-6-Glc]beta 1-3[PO4-6-Gal]beta 1-4Man, PO4-6Gal beta 1-3[PO4-6-Glc]beta 1-3Glc beta 1-3[PO4-6-Gal]beta 1-4Man, and PO4-6-Glc beta 1-3[PO4-6-Glc]beta 1-3Glc beta 1-3[PO4-6-Gal]beta 1-4Man. These glycans are linked together by the conserved phosphodiester R-Man alpha 1-PO4-6-Gal-R or the novel phosphodiester R-Man alpha 1-PO4-6-Glc-R and are connected to Ser(P) of the protein backbone most likely via the linkage R-Man alpha 1-PO4-Ser. The variety of stage-specific glycan structures in Leishmania mexicana aPPG suggests the presence of developmentally regulated amastigote glycosyltransferases which may be potential anti-parasite drug targets.

Hepatic structure-pharmacokinetic relationships: The hepatic disposition and metabolite kinetics of a homologous series of O-acyl derivatives of salicylic acid

1 The hepatic disposition and metabolite kinetics of a homologous series of O-acyl (acetyl, propionyl, butanoyl, pentanoyl, hexanoyl and octanoyl) esters of salicylic acid (C2SA, C3SA, C4SA, C5SA, C6SA and C8SA, respectively) was determined using a single-pass, in-sills rat liver preparation. 2 The hepatic venous outflow profiles for the parent esters and the generated metabolite, salicylic acid (SA) were analysed by HPLC. Non-parametric moments analysis was used to determine the area under the curve (AUC'), mean transit time (MTT) and normalized variance (CV2) for the parent esters and generated SA. 3 Pregenerated SA ([C-14]-salicylic acid) was injected into each liver with the parent ester to determine its distribution characteristics. 4 The overall recovery of ester plus metabolite was 89% of the ester dose injected and independent of the ester carbon number, suggesting that ester extraction was due to hepatic metabolism to salicylic acid. 5 The metabolite AUC' value increased directly with the lipophilicity of the parent ester (from 0.12 for C2SA to 0.95 for C8SA). By contrast, the parent AUC' decreased with the lipophilicity (from 0.85 for C2SA to zero for C8SA). The metabolite MTT value also showed a trend to increase with the lipophilicity of the parent ester (from 15.72 s for C3SA to 61.97 s for C8SA). However, the parent MTT value shows no significant change across the series. 6 The two-compartment dispersion model was used to derive the kinetic parameters for parent ester, pregenerated SA and generated SA. Consequently, these parameters were used to estimate the values of AUG', MITT and CV2 for the parent ester and metabolite. The moments values obtained using the two-compartment dispersion model show similar trends to the corresponding moments values obtained from the outflow profiles using a non-parametric approach. 7 The more lipophilic aspirin analogues are more confined to the portal circulation after oral administration than aspirin due to their more extensive hepatic elimination avoiding systemic prostacyclin inhibition. Given that aspirin's selectivity as an anti-thrombotic agent has been postulated to be due to selective anti-platelet effects in the portal circulation, the more lipophilic and highly extracted analogues are potentially more selective anti-thrombotic agents than aspirin.

Selection of a strain of Aspergillus for the production of citric acid from pineapple waste in solid-state fermentation

Aspergillus foetidus ACR I 3996 (=FRR 3558) and three strains of Aspergillus niger ACM 4992 (=ATCC 9142), ACM 4993 (=ATCC 10577), ACM 4994 (=ATCC 12846) were compared for the production of citric acid from pineapple peel in solid-state fermentation. A. niger ACM 4992 produced the highest amount of citric acid, with a yield of 19.4 g of citric acid per 100 g of dry fermented pineapple waste under optimum conditions, representing a yield of 0.74 g citric acid/g sugar consumed. Optimal conditions were 65% (w/w) initial moisture content, 3% (v/w) methanol, 30 degrees C, an unadjusted initial pH of 3.4, a particle size of 2 mm and 5 ppm Fe2+. Citric acid production was best in flasks, with lower yields being obtained in tray and rotating drum bioreactors.

Calcium-permeable AMPA receptors mediate long-term potentiation in interneurons in the amygdala

Fear conditioning is a paradigm that has been used as a model for emotional learning in animals'. The cellular correlate of fear conditioning is thought to be associative N-methyl-D-aspartate (NMDA) receptor-dependent synaptic plasticity within the amygdala(1-3). Here we show that glutamatergic synaptic transmission to inhibitory interneurons in the basolateral amygdala is mediated solely by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. In contrast to AMPA receptors at inputs to pyramidal neurons, these receptors have an inwardly rectifying current-voltage relationship, indicative of a high permeability to calcium(4 5), Tetanic stimulation of inputs to interneurons caused an immediate and sustained increase in the efficacy of these synapses. This potentiation required a rise in postsynaptic calcium, but was independent of NMDA receptor activation. The potentiation of excitatory inputs to interneurons was reflected as an increase in the amplitude of the GABAA-mediated inhibitory synaptic current in pyramidal neurons. These results demonstrate that excitatory synapses onto interneurons within a fear conditioning circuit show NMDA-receptor independent long-term potentiation. This plasticity might underlie the increased synchronization of activity between neurons in the basolateral amygdala after fear conditioning(6).

Retinoic acid disrupts anterior ectodermal and endodermal development in ascidian larvae and postlarvae

In vertebrates, excess all-trans retinoic acid (RA) applied during axis formation leads to the apparent truncation of anterior structures. In this study we sought to determine the type of defects caused by ectopic RA on the development of the ascidian Herdmania curvata. We demonstrate that H. curvata embryos cultured in the presence of RA develop into larvae whose trunks are shortened and superficially resemble those of early metamorphosing postlarvae. Despite RA-treated larvae lacking papillar structures they respond normally to natural cues that induce metamorphosis, indicating that chemosensory functionality previously mapped to the most anterior region of normal larvae is unaffected by RA. Excess RA applied during postlarval development leads to a graded loss of the juvenile pharynx, apparently by respecifying anterior endoderm to a more posterior fate. This structure is considered homologous to the gill slits of amphioxus. which are also lost upon RA treatment. This suggests that RA may have had a role in the development of the pharynx of the ancestral chordate and that this function has been maintained in ascidians and cephalochordates and lost in vertebrates.

Chronic ethanol treatment leads to increased ornithine decarboxylase activity: Implications for a role of polyamines in ethanol dependence and withdrawal

Recent research has focused on the N-methyl-D-aspartate receptor system as a major site of ethanol action in the brain and specifically on compensatory changes in the expression of the polyamine-sensitive NR2B subunit. Therefore, we examined the effects of chronic ethanol treatment on polyamine homeostasis in the rat brain. Wistar rats were made dependent by ethanol vapor inhalation. This caused a rise in hippocampal ornithine decarboxylase (ODC) activity that was correlated with the appearance of physiological dependence. ODC activity returned to control levels within 3 days of ethanol withdrawal. Enzyme activity also increased in the cerebral cortex, striatum, and cerebellum of the ethanol-dependent rats. The concentration of the polyamines (putrescine, spermidine, and spermine) in the hippocampus was increased in ethanol-dependent rats. Injection of the ODC inhibitor, gamma-difluoromethylornithine (500 mg/kg) at the onset of withdrawal resulted in a significant reduction in the severity of withdrawal behaviors. The level of ODC activity and the severity of withdrawal behaviors were positively correlated. Perturbed polyamine homeostasis may represent an important molecular component in the initiation of ethanol withdrawal behaviors in the ethanol-dependent rat.

Evaluation of an immunoassay (EMIT) for mycophenolic acid in plasma from renal transplant recipients compared with a high-performance liquid chromatography assay

Mycophenolic acid is an immunosuppressant administered as a bioavailable ester, mycophenolate mofetil. The pharmacokinetics of mycophenolic acid have been reported to be variable. Accurate measurement of concentrations of this drug could be important to adjust doses. The aim of this study was to compare the enzyme-multiplied immunoassay technique (EMIT [Dade Behring; San Jose, CA, U.S.A.]) for mycophenolic acid with a high-performance liquid chromatographic (HPLC) assay using samples collected from renal transplant recipients. The HPLC assay used solid phase extraction and a C18 stationary phase with ultraviolet (UV) detection (254 nm). The immunoassay required no manual sample preparation. Plasma samples (n = 102) from seven patients, collected at various times after a dose, were analyzed using both methods. Both assays fulfilled quality-control criteria. Higher concentrations were consistently measured in patient samples when using EMIT. The mean (+/- standard deviation [SD]) bias (EMIT-HPLC) was 1.88 +/- 0.86 mg/L. The differences in concentrations were higher in the middle of a dosage interval, suggesting that a metabolite might have been responsible for overestimation. Measurement of glucuronide concentrations by HPLC demonstrated only a weak correlation between assay differences and glucuronide concentrations. If the crossreacting substance is active, EMIT could provide a superior measure of immunosuppression; if inactive, further work is needed to improve antibody specificity. In conclusion, it was found that EMIT overestimates the concentration of mycophenolic acid in plasma samples from renal transplant recipients compared with HPLC analysis.

Crystallization of importin alpha, the nuclear-import receptor

Crystals of recombinant importin alpha, the nuclear-import receptor, have been obtained at two different pH conditions by vapour diffusion using sodium citrate as precipitant and dithiothreitol as an additive. At pH 4-5, the crystals have the symmetry of the trigonal space group P3(1)21 or P3(2)21 (a = b = 78.0, c = 255.8 Angstrom, gamma = 120 degrees); at pH 6-7, the crystals have the symmetry of the orthorhombic space group P2(1)2(1)2(1) (a = 78.5, b = 89.7, c = 100.5 Angstrom). In both cases, there is probably one molecule of importin ct in the asymmetric unit. At least one of the crystal forms diffracts to a resolution higher than 3 Angstrom using the laboratory X-ray source; the crystals are suitable for crystal structure determination.

Role of the 6-20 disulfide bridge in the structure and activity of epidermal growth factor

Two synthetic analogues of murine epidermal. growth factor, [Abu6, 20] mEGF4-48 (where Abu denotes amino-butyric acid) and [G1, M3, K21, H40] mEGF1-48, have been investigated by NMR spectroscopy. [Abu6, 20] mEGF4-48 was designed to determine the contribution of the 6-20 disulfide bridge to the structure and function of mEGF The overall structure of this analogue was similar to that of native mEGF, indicating that the loss of the 6-20 disulfide bridge did not affect the global fold of the molecule. Significant structural differences were observed near the N-terminus, however, with the direction of the polypeptide chain between residues four and nine being altered such that these residues were now located on the opposite face of the main beta-sheet from their position in native mEGF Thermal denaturation experiments also showed that the structure of [Abu6, 20] mEGF4-48 was less stable than that of mEGF. Removal of this disulfide bridge resulted in a significant loss of both mitogenic activity in Balb/c 3T3 cells and receptor binding on A431 cells compared with native mEGF and mEGF4-48, implying that the structural changes in [Abu6, 20] mEGF4-48, although limited to the N-terminus, were sufficient to interfere with receptor binding. The loss of binding affinity probably arose mainly from steric interactions of the dislocated N-terminal region with part of the receptor binding surface of EGF [G1, M3, K21, H40] mEGF1-48 was also synthesized in order to compare the synthetic polypeptide with the corresponding product of recombinant expression. Its mitogenic activity in Balb/c 3T3 cells was similar to that of native mEGF and analysis of its H-1 chemical shifts suggested that its structure was also very similar to native.

alpha-Conotoxin ImI inhibits the alpha-bungarotoxin-resistant nicotinic response in bovine adrenal chromaffin cells

The activity of alpha-conotoxin (alpha-CTX) lml, from the vermivorous marine snail Conus imperialis, has been studied on mammalian nicotinic receptors on bovine chromaffin cells and at the rat neuromuscular junction. Synthetic alpha-CTX lml was a potent inhibitor of the neuronal[ nicotinic response in bovine adrenal chromaffin cells (IC50 = 2.5 mu M, log IC50 = 0.4 +/- 0.07), showing competitive inhibition of nicotine-evoked catecholamine secretion. (alpha-CTX lml also inhibited nicotine-evoked Ca-45(2+) uptake but not Ca-45(2+) uptake stimulated by 56 mM Kr. In contrast, alpha-CTX lml had no effect at the neuromuscular junction over the concentration range 1-20 mu M. Bovine chromaffin cells are known to contain the alpha 3 beta 4, alpha 7, and (possibly) alpha 3 beta 4 alpha 5 subtypes. However, the secretory response of bovine chromaffin cells is not inhibited by alpha-bungarotoxin, indicating that alpha 7 nicotinic receptors are not involved. We propose that alpha-CTX lml interacts selectively with the functional (alpha 3 beta 4 or alpha 3 beta 4 alpha 5) nicotinic acetylcholine receptor to inhibit the neuronal-type nicotinic response in bovine chromaffin cells.