Intracellular polyamines mediate inward rectification of Ca(2+)-permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors.

alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors that lack the glutamate receptor GluR2 subunit are Ca(2+)-permeable and exhibit inwardly rectifying current responses to kainate and AMPA. A proportion of cultured rat hippocampal neurons show similar Ca(2+)-permeable inwardly rectifying AMPA receptor currents. Inward rectification in these neurons was lost with intracellular dialysis and was not present in excised outside-out patches but was maintained in perforated-patch whole-cell recordings, suggesting that a diffusible cytoplasmic factor may be responsible for rectification. Inclusion of the naturally occurring polyamines spermine and spermidine in the recording pipette prevented loss of rectification in both whole-cell and excised-patch recordings; Mg2+ and putrescine were without effect. Inward rectification of Ca(2+)-permeable AMPA receptors may reflect voltage-dependent channel block by intracellular polyamines.

Periodicity of polar and nonpolar amino acids is the major determinant of secondary structure in self-assembling oligomeric peptides.

The tendency of a polypeptide chain to form alpha-helical or beta-strand secondary structure depends upon local and nonlocal effects. Local effects reflect the intrinsic propensities of the amino acid residues for particular secondary structures, while nonlocal effects reflect the positioning of the individual residues in the context of the entire amino acid sequence. In particular, the periodicity of polar and nonpolar residues specifies whether a given sequence is consistent with amphiphilic alpha-helices or beta-strands. The importance of intrinsic propensities was compared to that of polar/nonpolar periodicity by a direct competition. Synthetic peptides were designed using residues with intrinsic propensities that favored one or the other type of secondary structure. The polar/nonpolar periodicities of the peptides were designed either to be consistent with the secondary structure favored by the intrinsic propensities of the component residues or in other cases to oppose these intrinsic propensities. Characterization of the synthetic peptides demonstrated that in all cases the observed secondary structure correlates with the periodicity of the peptide sequence--even when this secondary structure differs from that predicted from the intrinsic propensities of the component amino acids. The observed secondary structures are concentration dependent, indicating that oligomerization of the amphiphilic peptides is responsible for the observed secondary structures. Thus, for self-assembling oligomeric peptides, the polar/nonpolar periodicity can overwhelm the intrinsic propensities of the amino acid residues and serves as the major determinant of peptide secondary structure.

Organic properties of sediments and amino acids in iterstitial waters of ODP Holes 111-677A and 111-678B

The basement at Ocean Drilling Program (ODP) Sites 677 and 678 originated from the Galapagos spreading center of the Costa Rica Rift and has moved about 200 km over the last 6 m.y. (Fig. 1) (Shipboard Scientific Party, 1987, 1988; Scientific Drilling Party, 1987). Sediments about 300 m thick cover basement so young that basal sediments at Sites 677 and 678 have been reheated up to 60?-70?C at Site 677 and altered to limestone and/or chert (Shipboard Scientific Party, 1988). Sediments from both sites indicate (1) a high sedimentation rate (about 48 m/m.y.) and (2) biogenic silica and carbonate as the main constituents of sediments (Table 1) (Shipboard Scientific Party, 1988). Heatflow observations and measurements of interstitial water chemistry around the sites show that Site 677 is in a lower heatflow zone (166 mW/m**2; 1°12.14'N, 83°44.22'W) whereas Site 678 is located in a zone of higher heat flow (250 mW/m**2; 1°13.01'N, 83°43.39'W) (Langseth et al., 1988; Shipboard Scientific Party, 1988). In the flank hydrothermal systems, circulating solution is moving upward through the sedimentary column in zones of higher heat flow while it is moving downward in zones of lower heat flow (Anderson and Skilbeck, 1981). The chemistry of the interstitial waters is modified by several processes such as (1) diagenetic reactions and (2) advective and (3) diffusive transports of dissolved constituents. Analyses of Ca2+ and Mg2+ in interstitial waters from Sites 677 and 678 show that their profiles are mainly controlled by advective transport (Shipboard Scientific Party, 1988). In contrast, the interstitial-water profiles for NH4+, Si, and PO4[3-] are highly affected by reactions in the sediments. Site 677 offers a good opportunity to investigate amino acids in the interstitial waters because sediments of similar compositions have been deposited at constant rates of sedimentation. There are few previous works on amino acid distributions in interstitial waters (Henrichs and Parrington, 1979; Michaelis et al., 1982; Henrichs et al., 1984; Henrichs and Farrington, 1987; Ishizuka et al., 1988). In this chapter, we report (1) Rock-Eval analysis and (2) the composition of total hydrolyzable and dissolved free amino acids (THAA and DFAA, respectively) in the interstitial waters. Our objectives are to discuss (1) the possible origin of organic materials, (2) the characteristics of THAA and DFAA, and (3) their relationships in interstitial waters.

Boric acid catalyzed chemoselective esterification of alpha-hydroxycarboxylic acids

Boric acid catalyzes the selective esterification of alpha-hydroxycarboxylic acids without causing significant esterification to occur with other carboxylic acids. The procedure is simple, high-yielding, and applicable to the esterification of alpha-hydroxy carboxylates in the presence of other carboxylic acids including beta-hydroxyacids within the same molecule.

Mechanism of attenuation of muscle protein degradation induced by tumor necrosis factor-alpha and angiotensin II by beta-hydroxy-beta-methylbutyrate

Both tumor necrosis factor-alpha (TNF-alpha)/interferon-gamma (IFN-gamma) and angiotensin II (ANG II) induced an increase in total protein degradation in murine myotubes, which was completely attenuated by treatment with beta-hydroxy-beta-methylbutyrate (HMB; 50 microM). There was an increase in formation of reactive oxygen species (ROS) within 30 min, as well as an increase in the activity of both caspase-3 and -8, and both effects were attenuated by HMB. Moreover, inhibitors of caspase-3 and -8 completely attenuated both ROS formation and total protein degradation induced by TNF-alpha/IFN-gamma and ANG II. There was an increased autophosphorylation of double-stranded RNA-dependent protein kinase (PKR), which was attenuated by the specific caspase-3 and -8 inhibitors. Neither ROS formation or protein degradation occurred in myotubes expressing a catalytically inactive PKR variant, PKRDelta6, in response to TNF-alpha/IFN-gamma, compared with myotubes expressing wild-type PKR, although there was still activation of caspase-3 and -8. HMB also attenuated activation of PKR, suggesting that it was important in protein degradation. Formation of ROS was attenuated by rotenone, an inhibitor of the mitochondrial electron transport chain, nitro-l-arginine methyl ester, an inhibitor of nitric oxide synthase, and SB 203580, a specific inhibitor of p38 mitogen-activated protein kinase (p38 MAPK), which also attenuated total protein degradation. Activation of p38 MAPK by PKR provides the link to ROS formation. These results suggest that TNF-alpha/IFN-gamma and ANG II induce muscle protein degradation by a common signaling pathway, which is attenuated by HMB, and that this involves the initial activation of caspase-3 and -8, followed by autophosphorylation and activation of PKR, which then leads to increased ROS formation via activation of p38 MAPK. Increased ROS formation is known to induce protein degradation through the ubiquitin-proteasome pathway.

Contribution of transmembrane domain V amino acids to β1l-adrenoceptor activity and affinity

There are two binding sites on the β1-adrenoceptor (AR), β1H and β1L corresponding to high and low affinity binding sites respectively, which can be activated to cause cardiostimulation (reviewed Kaumann and Molenaar, 2008). Some β-blockers that block β1AR and β2ARs can activate β1LARs at higher concentrations than those required to cause blockade. The β2AR does not form a corresponding low affinity binding site (Baker et al 2002) and therefore we postulated that heterologous amino acids are responsible for the formation of β1LAR. Our aim was to investigate whether heterologous amino acids of transmembrane domain V (TMDV) of β1AR and β2ARs contribute to β1LAR. β1ARs, β2ARs and mutant β1ARs containing all (β1(β2TMDV)AR) or single amino acids of TMDV of the β2AR were prepared and stably expressed in Chinese Hamster Ovary cells. Concentration-effect curves for cyclicAMP accumulation were carried out for (-)-CGP12177 or (-)-isoprenaline in the absence or presence of (-)-bupranolol. _______________________________________________________________________ (-)-CGP 12177 (-)-Bupranolol affinity (pKB) pEC50 vs (-)-CGP 12177 vs (-)-isoprenaline _______________________________________________________________________ β1AR 8.00 ± 0.11 (11) 7.23 ± 0.23 (5) 9.52 ± 0.28 (5) β2AR (high density) 9.24 ± 0.14 (5) 9.82 ± 0.52 (8) xPaulxxxxxxx β2AR (low density) no effect β1(β2TMV)AR 8.86 ± 0.10 (15) 8.06 ± 0.17 (8) 9.08 ± 0.22 (6) β1(V230I)AR 9.07 ± 0.07 (10) 7.64 ± 0.12 (8) 9.36 ± 0.28 (9) β1(R222Q)AR 8.09 ± 0.29 (6) 7.33 ± 0.23 (5) 9.36 ± 0.08 (6) β1(V230A)AR 7.59 ± 0.09 (6) 7.32 ± 0.24 (4) 8.62 ± 0.18 (5) _______________________________________________________________________ The potency of (-)-CGP12177 was higher at β2AR than at β1AR consistent with activation through a low affinity site at the β1AR (β1LAR) but not β2AR. The presence of V230 in β1AR accounted for the lower potency of (-)-CGP 12177. The affinity of (-)-bupranolol at β1AR and mutants was higher when determined with (-)-isoprenaline than with (-)-CGP 12177. The affinity of (-)-bupranolol determined against (-)-CGP 12177 was lower at β1AR compared to β2AR. The presence of V230 in β1AR accounted in part for the lower affinity. In conclusion V230 of the β1AR contributes in part to the low affinity binding site of β1AR. Baker JG, Hall IP, Hill SJ (2002). Pharmacological characterization of CGP12177 at the human β2-adrenoceptor. Br J Pharmacol 137, 400−408 Kaumann AJ, Molenaar P (2008) The low-affinity site of the β1-adrenoceptor and its relevance to cardiovascular pharmacology. Pharmacol Ther 118, 303-336

Identification and characterization of a library of microheterogeneous cyclohexadepsipeptides from the fungus Isaria

Ten new cyclic hexadepsipeptides, six isariins and four isaridins, from the fungus Isaria have been identified and characterized by high-performance liquid chromatography, coupled to tandem electrospray ionization mass spectrometry (LC-ESIMS/MS). The isariins possess a beta-hydroxy acid residue and five alpha-amino acids, while isaridins contain a beta-amino acid, an alpha-hydroxy acid, and four alpha-amino acids. One- and two-dimensional NMR spectroscopy confirmed the chemical identity of some of the isariin fractions. Mass spectral fragmentation patterns of [M + H](+) ions reveal clear diagnostic fragment ions for the isariins and isaridins. Previously described cyclic depsipeptides, isarfelins from Isaria felina (Guo, Y. X.; Liu, Q. H.; Ng, T. B.; Wang H. X. Peptides 2005, 26, 2384), are now reassigned as members of the isaridin family. Examination of isaridin sequences revealed significant similarities with cyclic hexadepsipeptides such as destruxins and roseotoxins. The structure of an isariin (isariin A) investigated by NMR spectroscopy indicated the presence of a hybrid alpha beta C-11 turn, formed by the beta-hydroxy acid and glycine residues and a (D)Leu-(L)Ala type II' beta-turn. Additionally, the inhibitory effect of isariins and an isaridin on the intra-erythrocytic growth of Plasmodium falciparum is presented.

Identification and characterization of a library of microheterogeneous cyclohexa-depsipeptides from the fungus

Ten new cyclic hexadepsipeptides, six isariins and four isaridins, from the fungus Isaria have been identified and characterized by high-performance liquid chromatography, coupled to tandem electrospray ionization mass spectrometry (LC-ESIMS/MS). The isariins possess a beta-hydroxy acid residue and five alpha-amino acids, while isaridins contain a beta-amino acid, an alpha-hydroxy acid, and four alpha-amino acids. One- and two-dimensional NMR spectroscopy confirmed the chemical identity of some of the isariin fractions. Mass spectral fragmentation patterns of [M + H](+) ions reveal clear diagnostic fragment ions for the isariins and isaridins. Previously described cyclic depsipeptides, isarfelins from Isaria felina (Guo, Y. X.; Liu, Q. H.; Ng, T. B.; Wang H. X. Peptides 2005, 26, 2384), are now reassigned as members of the isaridin family. Examination of isaridin sequences revealed significant similarities with cyclic hexadepsipeptides such as destruxins and roseotoxins. The structure of an isariin (isariin A) investigated by NMR spectroscopy indicated the presence of a hybrid alpha beta C-11 turn, formed by the beta-hydroxy acid and glycine residues and a (D)Leu-(L)Ala type II' beta-turn. Additionally, the inhibitory effect of isariins and an isaridin on the intra-erythrocytic growth of Plasmodium falciparum is presented.

X-ray studies on crystalline complexes involving amino acids and peptides. XXV. Structures of DL-proline hemisuccinic acid and glycyl-L-histidinium semisuccinate monohydrate and a comparative study of amino-acid and peptide complexes of succinic acid

DL-Proline hemisuccinic acid, C5H9NO2.1/2C4H6O4, M(r) = 174.2, P2(1/c) a = 5.254 (1), b = 17.480 (1), c = 10.230 (i) angstrom, beta = 119.60 (6)-degrees Z = 4, D(m) = 1.41 (4), D(x) = 1.42 g cm-3, R = 0.045 for 973 observed reflections. Glycyl-L-histidinium semisuccinate monohydrate, C8H13N4O3+.C4H5O4-.H2O, M(r) = 348.4, P2(1), a = 4.864 (1), b = 17.071 (2), c = 9.397 (1) angstrom, beta = 90.58-degrees, Z = 2, D(m) = 1.45 (1), D(x) = 1.48 g cm-3, R = 0.027 for 1610 observed reflections. Normal amino-acid and dipeptide aggregation patterns are preserved in the structures in spite of the presence of succinic acid/semisuccinate ions. In both the structures, the amino-acid/dipeptide layers stack in such a way that the succinic acid molecules/semisuccinate ions are enclosed in voids created during stacking. Substantial variability in the ionization state and the stoichiometry is observed in amino-acid and peptide complexes of succinic acid. Succinic acid molecules and succinate ions appear to prefer a planar centro-symmetric conformation with the two carboxyl (carboxylate) groups trans with respect to the central C=C bond. Considerable variation is seen in the departure from and modification of normal amino-acid aggregation patterns produced by the presence of succinic acid. Some of the complexes can be described as inclusion compounds with the amino acid/dipeptide as the 'host' and succinic acid/semisuccinate/succinate as the 'guest'. The effects of change in chirality, though very substantial, are not the same in different pairs of complexes involving DL and L isomers of the same amino acid.

Hybrid Polypeptides: Gabapentin as a Stereochemically Constrained gamma-Amino Acid Residue

The design of folded structures in peptides containing the higher homologues of alpha-amino acid residues requires the restriction of the range of local conformational choices In alpha-amino acids stereochemically constrained residues like alpha,alpha-dialkylated residue, aminoisobutyric acid (Aib), and D-Proline ((D)Pro) have proved extremely useful in the design of helices and hairpins in short peptides Extending this approach, backbone substitution and cyclization are anticipated to bc useful in generating conformationally constrained beta- and gamma-residues This brief review provides a survey of work on hybrid peptide sequences concerning the conformationally constrained gamma-amino acid residue 1-aminomethyl cyclohexane acetic acid, gabapentin (Gpn) This achiral, beta,beta-disubstituted, gamma-residue strongly favors gauche-gauche conformations about the C-alpha-C-beta (0(2)) and C-alpha-C-gamma (0(1)) bonds, facilitating local folding The Gpn residue can adopt both C-7 (NH1 -> CO1) and C-9 (CO1 (I)<- NH1+I) hydrogen bonds which are analogous to the C-5 and C7 (gamma-turn) conformations at alpha-residues In conjunction with adjacent residues, Gpn may be used in ay and gamma alpha segments to generate C-12 hydrogen bonded conformations which may be considered as expanded analogs of conventional beta-turns The structural characterization of C-12 helices, C-12/C-10 helices with mixed hydrogen bond directionalities and beta-hairpins incorporating Gpn residues at the turn segment is illustrated (C) 2010 Wiley Periodicals, Inc Biopolymers (Pept Sci) 94 733-741 2010

Directing peptide conformation with centrally positioned pre-organized dipeptide segments: studies of a 12-residue helix and beta-hairpin

Secondary structure formation in oligopeptides can be induced by short nucleating segments with a high propensity to form hydrogen bonded turn conformations. Type I/III turns facilitate helical folding while type II'/I' turns favour hairpin formation. This principle is experimentally verified by studies of two designed dodecapeptides, Boc-Val-Phe-Leu-Phe-Val-Aib-Aib-Val-Phe-Leu-Phe-Val-OMe 1 and Boc-Val-Phe-Leu-Phe-Val- (D) Pro- (L) Pro-Val-Phe-Leu-Phe-Val-OMe 2. The N- and C-terminal flanking pentapeptide sequences in both cases are identical. Peptide 1 adopts a largely alpha-helical conformation in crystals, with a small 3(10) helical segment at the N-terminus. The overall helical fold is maintained in methanol solution as evidenced by NMR studies. Peptide 2 adopts an antiparallel beta-hairpin conformation stabilized by 6 interstrand hydrogen bonds. Key nuclear Overhauser effects (NOEs) provide evidence for the antiparallel beta-hairpin structure. Aromatic proton chemical shifts provide a clear distinction between the conformation of peptides 1 (helical) and 2 (beta-hairpin). The proximity of facing aromatic residues positioned at non-hydrogen bonding positions in the hairpin results in extensively ring current shifted proton resonances in peptide 2.

Systematic study of the transfer of amino acids across the water/1,2-dichloroethane interface facilitated by dibenzo-18-crown-6

Facilitated ion transfer reactions of 20 amino acids with di.benzo-18-crown-6 (DB18C6) at the water/1,2-dichloroethane (W/DCE) interfaces supported at the tips of micro- and nano-pipets were investigated systematically using cyclic voltammetry. It was found that there were only 10 amino acids, that is, Leu, Val, Ile, Phe, Trp, Met, Ala, Gly, Cys, Gln (in brief), whose protonated forms as cations can give well-defined facilitated ion transfer voltammograms within the potential window, and the reaction pathway was proven to be consistent with the transfer by interfacial complexation/dissociation (TIC/TID) mechanisms. The association constants of DB 18C6 with different amino acids in the DCE (beta(0)), and the kinetic parameters of reaction were evaluated based on the steady-state voltammetry of micro- or nano-pipets, respectively The experimental results demonstrated that the selectivity of complexation of protonated amino acid by DB18C6 compared with that of alkali metal cations was low, which can be attributed to the vicinal effect arising from steric hindrance introduced by their side group and the steric bulk effect by lipophilic stabilization.

Electrospray ionization mass spectrometry of cyclodextrin complexes with amino acids in incubated solutions and in eluates of gel permeation chromatography

1:1 complexes of beta-cyclodextrin (CD) with three amino acids (Gly, Phe and Trp) have been detected as ions in the gas phase using infusion positive and negative ion electrospray ionization mass spectrometry (ESI-MS). In contrast with the positive ion ESI mass spectra of simple aqueous solutions, the aggregates and adducts usually formed in the ESI process did not appear in the positive ion ESI spectra of solutions buffered with ammonium acetate (NH4Ac), even at higher analyte concentrations, These studies suggest that addition of buffer and/or use of a low analyte concentration should be used to overcome formation of aggregates and metal ion adducts in such mass spectrometry studies. Also, the deprotonated complexes are dissociated by collision induced dissociation (CID) to form an abundant product ion, the deprotonated CD, requiring transfer of a proton to the amino acid carboxyl group, To understand formation of complexes in the gas phase, gel permeation chromatography (GPC) was used to separate free amino acids (AAs) from complexes in an incubated solution. The ESI mass spectra of the GPC fractions show the presence of 1:1 complexes of both CD-aromatic amino acids and CD-aliphatic amino acids. Compared with CD-aliphatic amino acid complexes, CD-aromatic amino acid complexes appear to be destabilized in the gas phase, possibly because the hydrophobic interaction which binds the aromatic group of amino acids in the CD cavity in solution may become repulsive when solvent evaporates from the droplets during the electrospray process, whereas those complex ions formed as proton bound dimers are stabilized by electrostatic forces, the major binding force for such complexes in the gas phase. In addition, the GPC technique coupled with off-line ESI-MS can rapidly separate CD complexes by size, and provides some information on the character of the complexes in solution. (C) 1998 John Wiley & Sons, Ltd.