p-cresol as a reversible acylium ion scavenger in solid-phase peptide synthesis

In this work we have defined the nature of the p-cresol and p-thiocresol adducts generated from acylium ions during HF cleavage, following contemporary Boc/benzyl solid-phase peptide synthesis. Contrary to the results in previous reports, we found that both p-cresol and p-thiocresol predominantly form. aryl esters under typical cleavage conditions. Initially we investigated a number of small peptides containing either a single glutamate residue or a C-terminal long-chain amino acid which allowed us to unambiguously characterize the scavenged side products. Whereas, the p-cresol esters are stable at 0 degrees C they rearrange irreversibly at higher temperatures (5-20 degrees C) to form aryl ketones. By contrast, p-thiocresol esters do not undergo a Fries rearrangement but readily undergo further additions of p-thiocresol to form ketenebisthioacetals and trithio ortho esters, even at low temperatures. Importantly, we found by LC/MS and FT-ICR MS analysis that peptides containing p-cresol esters at glutamyl side chains are susceptible to amidation and fragmentation reactions at these sites during standard mild base workup procedures. The significance of these side reactions was further demonstrated in the synthesis of neutrophil immobilization factor, a 26-residue peptide, containing four glutamic acid residues. The side reactions were largely avoided by mild hydrogen peroxide-catalyzed hydrolysis which converted the p-cresol adducts to the free carboxylic acids in near quantitative yield. The choice of p-cresol as a reversible acylium ion scavenger when coupled with the simple workup conditions described is broadly applicable to Boc/benzyl peptide synthesis and will significantly enhance the quality of peptides produced.

Amino acid synthesis in the symbiotic sea anemone Aiptasia pulchella

Symbiotic Aiptasia pulchella and freshly isolated zooxanthellae were incubated in (NaHCO3)-C-14 and NH4Cl for 1 to 240 min, and samples were analysed by reverse-phase high-performance liquid chromatography (HPLC) and an online radiochemical detector. NH4+ was first assimilated into C-14-glutamate and C-14-glutamine in the zooxanthellae residing in A. pulchella. The specific activities (dpm nmol(-1)) of C-14-glutamate and C-14-glutamine in vivo, were far greater in the zooxanthellae than in the host tissue, indicating that NH4+ was principally incorporated into the glutamate and glutamine pools of the zooxanthellae. C-14-alpha-ketoglutarate was taken up from the medium by intact A. pulchella and assimilated into a small amount of C-14-glutamate in the host tissue, but no C-14-glutamine was detected in the host fraction. The C-14-glutamate that was synthesized was most likely produced from transamination reactions as opposed to the direct assimilation of NH4+. The free aminoacid composition of the host tissue and zooxanthellae of A. pulchella was also measured. The results presented here demonstrate that NH4+ was initially assimilated by the zooxanthellae of A. pulchella.

Comparative in vitro effects of closantel and selected beta-ketoamide anthelmintics on a gastrointestinal nematode and vertebrate liver cells

PNU-87407 and PrNU-88509, beta-ketoamide anthelmintics that are structurally related to each other and to the salicylanilide anthelmintic closantel, exhibit different anthelmintic spectra and apparent toxicity in mammals, The basis for this differential pharmacology was examined in experiments that measured motility and adenosine triphosphate (ATP) levels in larval and adult stages of the gastrointestinal nematode, Haemonchus contortus, and in a vertebrate liver cell line and mitochondria, PNU-87407 and PNU-88509 both exhibited functional cross-resistance with closantel in larval migration assays using closantel-resistant and -sensitive isolates of H, contortus. Each compound reduced motility and,ATP levels in cultured adult H. contortus in a concentration- and time-dependent manner: however, motility was reduced more rapidly by PNU-88509, and ATP levels were reduced by lower concentrations of closantel than the beta-ketoamides. Tension recordings from segments of adult H, contortus showed that PNU-88509 induces spastic paralysis, while PNU-87407 and closantel induce flaccid paralysis of the somatic musculature. Marked differences in the actions of these compounds were also observed in the mammalian preparations. In Chang liver cells, ATP levels were reduced after 3 h exposures to greater than or equal to 0.25 mu M PNU-87407 1 mu M closantel or 10 mu M PNU-88509, Reductions in ATP caused by PNU-88509 were completely reversible, while the effects of closantel and PNU-87407; were irreversible. PNU-87407, closantel and PNU-88509 uncoupled oxidative phosphorylation in isolated rat liver mitochondria, inhibiting the respiratory control index (with glutamate or succinate as substrate) by 50% at concentrations of 0.14, 0.9 and 7.6 mu M respectively.

Presynaptic long-term depression at a central glutamatergic synapse: a role for CaMKII

CaMKII is a calcium-activated kinase that is abundant in neurons and has been strongly implicated in memory and learning. Here we show that low-frequency stimulation of glutamatergic afferents in hippocampal slices from juvenile domestic chicks results in long-term depression of synaptic transmission. This reduction does not require activation of NMDA or metabotropic glutamate receptors and does not require a rise in postsynaptic calcium. However, buffering presynaptic calcium prevents the reduction of the excitatory postsynaptic potential or current that is induced by low-frequency stimulation. in addition, application of the calmodulin antagonist calmidazolium, or the specific CaMKII antagonist KN-93, completely blocks long-term depression. These findings demonstrate a newsy discovered form of long-term synaptic depression in the avian hippocampus.

Derivatives of 1,3,5-triamino-1,3,5-trideoxy-cis-inositas versatile pentadentate ligands for protein labeling with Re-186/188. Prelabeling, biodistribution, and X-ray structural studies

The pentadentate H(3)bhci [1,3,5-trideoxy-1,3-bis((2-hydroxybenzyl)amino)-cis-inistol] and its bifunctionalized analogue H(3)bhci-glu-H [1,3,5-trideoxy-1,3-bis((2-hydroxybenzyl)amino)-5-glutaramido-cis-inositol] were synthesized, and their coordination chemistry was investigated with inactive rhenium, with no carrier added Re-188 and with carrier added Re-186. The neutral Re(V) complexes [ReO-(bhci)] and [ReO(bhci-glu-H)] are formed in good yields starting from [ReOCl3(P(C6H5)(3))(2)] or in quantitative yield directly from [(ReO4)-Re-186/188](-) in aqueous solution by reduction with Sn(II) or Sn(0). The X-ray structures of [ReO(bhci)] and [ReO(bhci-glu-H)] were elucidated revealing pentadentate side on coordination of the ligands to the Re=O core. The basic cyclohexane frame adopts a chair form in the case of [ReO(bhci)] and a twisted boat form in the case of [ReO(bhci-glu-H)]. [ReO(bhci)] crystallizes in the monoclinic space group C2/c with a = 27.425(3), b = 14.185(1), c = 19.047(2) Angstrom, and beta = 103.64(2)degrees and [ReO(bhci-glu-H)] in the monoclinic space group P2(1)/c with a = 13.056(3), b = 10.180(1), c = 22.378(5) Angstrom and beta = 98.205(9)degrees Both Re-188 complexes are stable in human serum for at least 3 days without decomposition. After injection into mice, [ReO(bhci-glu)](-) is readily excreted through the intestines, while [ReO(bhci)] is excreted by intestines, liver, and the kidneys. TLC investigations of the urine showed exclusively the complexes [ReO(bhci-glu-H)] and [ReO(bhci)], respectively, and no decomposition products. For derivatization of antibodies, the carboxylic group of [ReO(bhci-glu-H)] was activated with N-hydroxysuccinimide, which required unusually vigorous reaction conditions (heating). The anti colon cancer antibody mAb-35 [IgG and F(ab')(2) fragment] was labeled with [(ReO)-Re-186/188(bhci-glu)] to a specific activity of up to 1.5 mCi/mg (55 MBq/mg) with full retention of immunoreactivity. Labeling yields followed pseudo-first-order kinetics in antibody concentration with the ratio of rates between aminolysis and hydrolysis being about 2. Biodistributions of Re-186-labeled intact mAb-35 as well as of its F(ab')(2) fragment in tumor-bearing nude mice revealed good uptake by the tumor with only low accumulation of radioactivity in normal tissue.

Structure-activity studies of conantokins as human N-methyl-D-aspartate receptor modulators

The activities of conantokin-G (con-G), conantokin-T (con-T), and several novel analogues have been studied using polyamine enhancement of [H-3]MK-801 binding to human glutamate-N-methyl-D-aspartate (NMDA) receptors, and their structures have been examined using CD and H-1 NMR spectroscopy. The potencies of con-G[A7], con-G, and con-T as noncompetitive inhibitors of spermine-enhanced [H-3]MK-801 binding to NMDA receptor obtained from human brain tissue are similar to those obtained using rat brain tissue. The secondary structure and activity of con-G are found to be highly sensitive to amino acid substitution and modification. NMR chemical shift data indicate that con-G, con-G[D8,D17], and con-G[A7] have similar conformations in the presence of Ca2+. This consists of a helix for residues 2-16, which is kinked in the vicinity of Gla10. This is confirmed by 3D structure calculations on con-G[A7]. Restraining this helix in a linear form (i.e., con-G[A7,E10-K13]) results in a minor reduction in potency. Incorporation of a 7-10 salt-bridge replacement (con-G[K7-E10]) prevents helix formation in aqueous solution and produces a peptide with low potency. Peptides with the Leu5-Tyr5 substitution also have low potencies (con-G[Y5,A7] and con-G[Y5,K7]) indicating that Leu5 in con-G is important for full antagonist behavior. We have also shown that the Gla-Ala7 substitution increases potency, whereas the Gla-Lys7 substitution has no effect. Con-G and con-G[K7] both exhibit selectivity between NMDA subtypes from mid-frontal and superior temporal gyri, but not between sensorimotor and mid-frontal gyri. Asn8 and/or Asn17 appear to be important for the ability of con-G to function as an inhibitor of polyamine-stimulated [3H]MK-801 binding, but not in maintaining secondary structure. The presence of Ca2+ does not increase the potencies of con-G and con-T for NMDA receptors but does stabilize the helical structures of con-G, con-G[D8,D17], and, to a lesser extent, con-G[A7]. The NMR data support the existence of at least two independent Ca2+-chelating sites in con-G, one involving Gla7 and possibly Gla3 and the other likely to involve Gla10 and/or Gla14.

The influence of Lys68 in decapeptide agonists of C5a on C5a receptor binding, activation and selectivity

The potent, conformationally biased C5a agonist peptide YSFKPMPLaR (C5a(65-74), Y65, F67, P69, P71, D-Ala73) was used as a template to gain insight into the nature and importance of lysine at position 68 in the peptide-receptor interaction. A panel of YSFKPMPLaR analogs with systematic substitutions for Lys68 was evaluated for C5a receptor (C5aR) binding affinity and activation in two well-characterized assay systems: human polymorphonuclear leukocytes (PMNs) and human fetal artery. In addition, we determined the activity of these new analogs in transfected rat basophilic leukemia (RBL) cells in which the Glu at position 199 of the C5aR (wtGlu199) was replaced by a Gin (C5aR-Gln199) or a Lys (C5aR-Lys199). Our results indicated that Lys68 in YSFKPMPLaR plays an important role in binding the C5aR expressed on PMNs and RBL cells. Furthermore, the data indicated that Lys68 interacted with Glu199 of the C5aR in PMNs and RBL cells. In human fetal artery, however, Lys68 substitutions had little or no effect on activity, which suggested that the receptor conformation may be different in this tissue. Thus, the interaction between Lys68 of the decapeptide agonist and Glu199 of the C5aR may be cell type-specific and may form the molecular basis for tissue-specific responses to C5a agonists.

Excitatory synaptic inputs to pyramidal neurons of the lateral amygdala

Whole-cell patch clamp recordings were made from pyramidal neurons in the rat lateral amygdala (LA). Synaptic currents were evoked by stimulating in either the external capsule (ec), internal capsule (ic) or basolateral nucleus (BLA). Stimulation of either the ic, ec or BLA evoked a glutamatergic excitatory synaptic current (EPSC) which was mediated by both non-NMDA and NMDA (N-methyl-D-aspartic acid) receptors, The ratio of the amplitude of the NMDA receptor-mediated component measured at +40 mV to the amplitude of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) component measured at -60 mV was similar regardless of whether EPSCs were evoked in the ec, ic or BLA. At resting membrane potentials, excitatory synaptic potentials evoked from either the ec or putative thalamic inputs were unaffected by application of the NMDA receptor antagonist APV. Spontaneous glutamatergic currents had two components to their decay phase. The slow component was selectively blocked by the NMDA receptor antagonist D-APV, indicating that AMPA and NMDA receptors are colocalized in spiny neurons. We conclude that pyramidal cells of the LA receive convergent inputs from the cortex, thalamus and basal nuclei. At all inputs, both AMPA/kainate and NMDA-type receptors are active and colocalized in the postsynaptic density.

The novel mitochondrial gene arrangement of the cattle tick, Boophilus microplus: Fivefold tandem repetition of a coding region

We sequenced across all of the gene boundaries in the mitochondrial genome of the cattle tick, Boophilus microplus, to determine the arrangement of its genes. The mtDNA of B. microplus has a coding region, composed of tRNA(Glu) and 60 bp of the 3' end of ND1, that is repeated five times. Boophilus microplus is the first coelomate animal known to have more than two copies of a coding sequence. The mitochondrial genome of B, microplus has other unusual features, including (1) reduced T arms in tRNAs, (2) an AT bias in codon use, (3) two control regions that have evolved in concert, (4) three gene rearrangements, and (5) a stem-loop between tRNA(Gln) and tRNA(Phe). The short T arms and small control regions (CRs) of B. microplus and other ticks suggest strong selection for small genomes. Imprecise termination of replication beyond its origin, which can account for the evolution of tandem repeats of coding regions in other mitochondrial genomes, cannot explain the evolution of the fivefold repeated sequence in the mitochondrial genome of B. microplus. Instead, slipped-strand mispairing or recombination are the most plausible explanations for the evolution of these tandem repeats.

Structure of a putative ancestral protein encoded by a single sequence repeat from a multidomain proteinase inhibitor gene from Nicotiana alata

Background: The ornamental tobacco Nicotiana alata produces a series of proteinase inhibitors (Pls) that are derived from a 43 kDa precursor protein, NaProPl. NaProPl contains six highly homologous repeats that fold to generate six separate structural domains, each corresponding to one of the native Pls. An unusual feature of NaProPl is that the structural domains lie across adjacent repeats and that the sixth Pl domain is generated from fragments of the first and sixth repeats. Although the homology of the repeats suggests that they may have arisen from gene duplication, the observed folding does not appear to support this. This study of the solution structure of a single NaProPl repeat (aPl1) forms a basis for unravelling the mechanism by which this protein may have evolved, Results: The three-dimensional structure of aPl1 closely resembles the triple-stranded antiparallel beta sheet observed in each of the native Pls. The five-residue sequence Glu-Glu-Lys-Lys-Asn, which forms the linker between the six structural domains in NaProPl, exists as a disordered loop in aPl1. The presence of this loop in aPl1 results in a loss of the characteristically flat and disc-like topography of the native inhibitors. Conclusions: A single repeat from NaProPl is capable of folding into a compact globular domain that displays native-like Pl activity. Consequently, it is possible that a similar single-domain inhibitor represents the ancestral protein from which NaProPl evolved.

Rectification of the olfactory cyclic nucleotide-gated channel by intracellular polyamines

Polyamine-induced inward rectification of cyclic nucleotide-gated channels was studied in inside-out patches from rat olfactory neurons. The polyamines, spermine, spermidine and putrescine, induced an 'instantaneous' voltage-dependent inhibition with K-d values at 0 mV of 39, 121 mu M and 2.7 mM, respectively. Hill coefficients for inhibition were significantly < 1, suggesting an allosteric inhibitory mechanism. The Woodhull model for voltage-dependent block predicted that all 3 polyamines bound to a site 1/3 of the electrical distance through the membrane from the internal side. Instantaneous inhibition was relieved at positive potentials, implying significant polyamine permeation. Spermine also induced exponential current relaxations to a 'steady-state' impermeant level. This inhibition was also mediated by a binding site 1/3 of the electrical distance through the pore, but with a K-d of 2.6 mM. Spermine inhibition was explained by postulating two spermine binding sites at a similar depth. Occupation of the first site occurs rapidly and with high affinity, but once a spermine molecule has bound, it inhibits spermine occupation of the second binding site via electrostatic repulsion. This repulsion is overcome at higher membrane potentials, but results in a lower apparent binding affinity for the second spermine molecule. The on-rate constant for the second spermine binding saturated at a low rate (similar to 200 sec(-1) at +120 mV), providing further evidence for an allosteric mechanism. Polyamine-induced inward rectification was significant at physiological concentrations.