Critical role of methionine-722 in the stimulation of human brain G-proteins and neurotoxicity induced by London familial Alzheimer's disease (FAD) mutated V717G-APP(714-723).

We have demonstrated earlier that V717G-APP(714-723), the membrane fragment of the V717G ("London") familial Alzheimer's disease (FAD) mutant of amyloid precursor protein (APP), is a potent stimulator of G-proteins in human brain membranes. In this study, we tested the hypothesis that Met-722 in the V717G-APP(714-723) peptide (P2) plays a critical role in the P2-induced oxidative stimulation of G-proteins in the human temporal cortex membranes and in the neurotoxicity of the peptide in differentiated PC12 and cerebellar granular cells. We found that 10 microM P3, the Met-722 sulfoxide analog of P2, produced a twofold lower stimulation of G-proteins ([(35)S]-GTPgammaS binding) in control temporal cortex membranes compared with 10 microM P2. The stimulatory effect of 10 microM P4, the Met-722 sulfone analog of P2, was 2.5-fold lower than the effect of P2. In Alzheimer's disease (AD) temporal cortex, the P3 and P4 stimulation of G-proteins was slightly weaker than the P2 stimulation. Substitution of the Met-722 S-atom in P2 by -CH(2)- group (P5) led to the disappearance of P2 stimulatory effect on G-proteins. Glutathione (GSH), melatonin (Mel), desferrioxamine (DFO) and 17-beta-estradiol (17betaE) significantly reduced P2 stimulatory effect on G-proteins in human brain. Only DFO and Mel were able to reduce the moderate stimulation of G-proteins by P3, whereas none of the tested antioxidants influenced the weak stimulation by P4. P2 at 100 microM induced a 40% decrease in PC12 cell viability as revealed by MTT assay, the effect being significantly higher than that of P3 or P4, whereas P1 (wild-type APP(714-723)) did not affect cell viability. Trypan Blue exclusion assay demonstrated that 10 microM P2 and P3 induced 3.8- and 3.5-fold death in the cerebellar granular cells as compared with the respective control values. P1 and P4 at 10 microM induced 1.7- and 2.3-fold increase in cell death, respectively. Treatment of the cerebellar granular cells with pertussis toxin decreased the high neurotoxicity of P2 and P3, whereas the low toxicity of P1 and P4 was not influenced. These results support the hypothesis that the G-protein stimulatory effect and neurotoxicity of "London"-mutated V717G-APP(714-723) (P2) and its Met-722 oxidized analogs involve oxidative-dependent and oxidative-independent mechanisms and the oxidation state of Met-722 plays a critical role in determining the mechanism.

PNA oligomers as tools for specific modulation of gene expression.

Small synthetic molecules that can specifically inhibit translation and/or transcription have shown great promise as potential antisense/antigene drugs. Peptide nucleic acid (PNA), an oligonucleotide mimic, has a non-charged achiral polyamide backbone to which the nucleobases are attached. PNA oligomers are extremely stable in biological fluids and they specifically hybridise to DNA or RNA in a complementary manner, forming very strong heteroduplexes. Some of the mRNAs have yet undetermined and possibly long half-lives, successful down regulation of gene expression by antisense oligonucleotides (ON) requires that the antisense agent is long lived. PNA fulfils this requirement better than phosphodiester or phosphorothioate ONs. PNA can inhibit transcription and translation of respective genes by tight binding to DNA or mRNA. First in vitro experiments to specifically down regulate protein expression by PNA have been followed by successful antisense and antigene application of PNA oligomers in vivo. This review discusses the principles of the in vitro and in vivo use of PNA oligonucleotides.

Intrathecal administration of PNA targeting galanin receptor reduces galanin-mediated inhibitory effect in the rat spinal cord.

Peptide nucleic acids (PNA) are nucleic acid analogues containing neutral amide backbone, forming stable and tight complexes with complementary DNA/RNA. However, it is unclear whether unmodified PNA can efficiently penetrate neuronal tissue in order to act as antisense reagent. Here we show that intrathecal (i.t.) injection of an unmodified antisense PNA complementary to the rat galanin receptor type 1 (GalR1) mRNA is able to block the inhibitory effect of i.t. administered galanin on spinal nociceptive transmission. Autoradiographic ligand binding studies using [125I]galanin show that the unmodified PNA is able to reduce the density of galanin binding sites in the dorsal horn. Thus, unmodified PNA applied i.t. appears to function as an effective antisense reagent in rat spinal cord in vivo.

Hypothalamic degradation of galanin(1-29) and galanin(1-16): identification and characterization of the peptidolytic products.

The degradation of the neuropeptide galanin(1-29) and its fully active synthetic N-terminal fragment galanin(1-16) in hypothalamic tissue, where these peptides potently affect feeding behaviour, is studied. Galanin(1-29) had a half-life of 100 min while galanin(1-16) had a half-life of 28 min when incubated with a hypothalamic membrane preparation. The putative sites of peptidolytic cleavage of the active N-terminal fragment galanin(1-16) were determined as being between amino acids Leu4 and Asn5, between Asn5 and Ser6, and between His14 and Ala15, respectively. The synthetic analogs of galanin(1-16) where Leu4, Asn5 or Ser6 was substituted by Ala were all more stable to peptidolysis; [Ala4]galanin(1-16) had a half-life of 55 min. Cleavage of the galanin(1-16) between His14-Ala15 yields a ligand-galanin(1-14) which binds to the receptor with high affinity (KD approximately 10(7) M), while cleavage at amino acid residues Leu4, Asn5 and Ser6 results in inactive peptide fragments with affinities for the galanin receptor below 10(-4) M. The enzyme(s) responsible for degradation of galanin were identified as endopeptidase(s), which were partially inhibited by bacitracin (1 mg/ml) by up to 50%, but not significantly by EDTA (1 mM), phosphoramidon (1 microM), phenylmethylsulfonyl fluoride, (100 microM) or aprotinin (10 micrograms/ml).

Anatomical study of the rabbit's corneal-VIth nerve reflex: connections between cornea, trigeminal sensory complex, and the abducens and accessory abducens nuclei.

The corneal-VIth nerve reflex of the rabbit, involving retraction of the eyeball by the retractor bulbi muscle and the correlated extension of the nictitating membrane, has been suggested to be mediated by retractor bulbi motoneurons in the accessory abducens-(ACC) nucleus but not by those in the abducens (ABD) nucleus, and to consist of both a fast, disynaptic, component and a slower component mediated by the reticular formation (RF). We, therefore, employed the anterograde and retrograde transport of horseradish peroxidase (HRP) to examine the neural connections between anatomical structures proposed to be involved in the afferent limb of the corneal VIth nerve reflex. The transganglionic transport of HRP from cornea indicated a primary projection to the ventral half of pars oralis of the trigeminal sensory complex. The retrograde transport of HRP infused into ACC resulted in a bilateral labeling of cells in ventral pars oralis with 75% of the labeled cells being ipsilateral to the side of infusion. In contrast, there was no retrograde labeling of cells in the trigeminal sensory complex after HRP infusions into ABD. Infusion of HRP into ACC and ABD also revealed retrogradely labeled cells in the RF caudal to these two nuclei and infusion of HRP into this area of the RF resulted in both the retrograde labeling of cells in ventral pars oralis and anterograde-like labeling in both ACC and ABD. These data provide anatomical support for a direct relationship of the ACC, but not ABD, to the trigeminal sensory system and for the suggested existence of two components of the corneal-VIth nerve reflex: a disynaptic component from cornea to ventral pars oralis which in turn projects only to the ACC nucleus; and a multisynaptic component consisting of projections from the ventral pars oralis to RF cells which, in turn, are premotor to the ACC and ABD nuclei.

N-terminal galanin fragments inhibit the hippocampal release of acetylcholine in vivo.

Using synthetic N-terminal fragments of galanin, galanin (1-7), galanin (1-9), galanin (1-12) and galanin (1-16), we have shown that the minimal sequence required for inhibition of acetylcholine release in vivo from rat ventral hippocampus corresponds to galanin (1-12). The fragment (1-9) displays activity in vivo but only at a very high concentration of 6.23 nmol while galanin (1-7) and C-terminal fragment (17-29) are without effect. Binding studies showed that galanin (1-16) and galanin (1-12) bind with submicromolar IC50 values to rat hippocampal galanin receptors. Galanin (1-9) has substantially lower affinity towards rat ventral hippocampal galanin receptor.

Galanin receptors from human pituitary tumors assayed with human galanin as ligand.

Galanin (i.v.) elevates circulating growth hormone levels in humans, and human growth hormone producing tumors show galanin-like immunoreactivity. We have therefore investigated the presence of galanin receptors in sixteen human pituitary tumors. Specific binding of [125I]monoiodo-[Tyr26]-porcine galanin was found in membranes from four clinically inactive and three growth hormone producing tumors. The affinity of human, rat and porcine galanin to these receptor sites was identical (Kd = 0.9 nM). The rank order of potency of galanin receptor ligands was the same in the human pituitary tissue as in the rat and porcine pituitary, hypothalamus, pancreas and hippocampus. GTP (1 mM) or GMPP(NH)P (0.5 mM) lowered the apparent specific binding of [125I]galanin (0.2 nM), suggesting that the human pituitary galanin receptor is coupled via a G-protein similarly to galanin receptors in mouse, rat and pig. The possible significance of galanin receptors in pituitary tumors is discussed.

Differential effects of the putative galanin receptor antagonists M15 and M35 on striatal acetylcholine release.

The putative galanin receptor antagonists M15 and M35 were examined for their effects on the basal and the galanin-evoked release of acetylcholine in the striatum. Extracellular concentrations of acetylcholine were measured in male rats using in vivo microdialysis and high pressure liquid chromatography techniques. Galanin (300 microM or 3 nmol/10 microliters), perfused through the microdialysis membrane into the striatum, enhanced (100% increase) basal acetylcholine release. M35 (300 microM or 3 nmol/10 microliters) also stimulated the basal acetylcholine release to some extent (about 50%) while M15 at the same concentration failed to do so. When M15 and M35 were coinfused with galanin, the galanin-evoked acetylcholine release was blocked completely by M15 (300 microM or 3 nmol/10 microliters) but only partially by M35 (300 microM or 3 nmol/10 microliters). The increase in acetylcholine release induced by M35 (300 microM) was blocked by M15 (300 microM). It is concluded that M15 is a full galanin receptor antagonist while M35 behaves as a mixed agonist-antagonist in vivo in the rat striatum. Both M15 and M35 fully displaced 0.2 nM [125I]galanin from its binding sites in the striatal membranes. The Hill coefficient of these [125I]galanin displacement curves with M15 and M35 was 0.4-0.5 compared to unity in the case of galanin. Analysis of the displacement curves suggested that both M15 and M35 recognized two classes of galanin binding sites in striatal membranes of the rat. To explain the difference between M15 and M35 it is suggested that there may exist a putative subtype of galanin receptor in the striatum, which is differentially affected by M15 and M35.

The novel high-affinity antagonist, galantide, blocks the galanin-mediated inhibition of glucose-induced insulin secretion.

This study describes the synthesis and effects of the first antagonist to the widely distributed neuropeptide, galanin, which inhibits the secretion of insulin. The first galanin antagonist is a 20-amino acid-long chimeric peptide of the composition galanin-(1-12)-Pro-substance P-(5-11) amide: Gly-Trp-Thr-Leu-Asn-Ser-Ala-Gly-Tyr-Leu-Leu-Gly-Pro-Gln-Gln-Phe-Phe-Gly- Leu-Met amide. The peptide dose dependently (IC50 = 1.0 nM) antagonizes the galanin-mediated inhibition of the glucose-induced insulin secretion from mouse pancreatic islets. The antagonist was also found to displace 125I-monoiodo-[Tyr26]galanin from membranes of the insulin producing Rin m 5F cells with an IC50 value of less than 0.1 nM. The antagonist is named galantide.

Blockade of galanin-induced inhibition of insulin secretion from isolated mouse islets by the non-methionine containing antagonist M35.

The neuropeptide galanin occurs in pancreatic adrenergic nerves and has been suggested to be the adrenergic mediator of the stress-induced inhibition of insulin release. To study its physiological function, we recently synthesized a galanin-like galanin receptor antagonist, galantide. However, this antagonist contains a methionine moiety, and is therefore easily oxidized. We have now synthesized another galanin antagonist which does not contain methionine. This peptide, M35, is a chimeric 21 amino acid peptide in which galanin-(1-13) is coupled to bradykinin-(2-9). M35 (10 microM to 1 pM) had no effect by itself on glucose (11.1 mM)-stimulated insulin secretion in isolated mouse islets, but potently counteracted the inhibitory action of galanin (100 nM). The lowest effective dose of M35 was 10 nM. M35 did not counteract the inhibitory action of clonidine (1 microM) or somatostatin (1 microM) on insulin secretion. Furthermore, M35 displaced 125I-monoiodo-[Tyr26]galanin from membranes of insulin producing RINm5F cells. The displacement curve fitted to a two-site model in which 60% of label bound with a K1 of 0.1 +/- 0.01 nM and 40% with a K2 of 3 +/- 0.5 nM. In conclusion, M35 is a specific, non-methionine-containing galanin receptor antagonist on insulin-producing cells.

cDNA sequence, ligand biding, and regulation of galanin/GMAP in mouse brain.

The cDNA encoding the 29 amino acid-long neuropeptide galanin and its flanking peptide galanin message associated peptide (GMAP), has been cloned and sequenced from mouse hypothalamic cDNA. The primary sequence of mouse galanin is GWTLNSAGYLLGPHAIDNHRSFSDKHGLT, followed by an amidation signal GKR. There are now 12 galanin sequences known: human, porcine, dog, rat, bovine, chicken, sheep, alligator, bowfin, dogfish, trout and mouse. The N-terminal 14 amino acids are identical in all of these species and the whole primary sequence of mouse galanin is identical to that of rate galanin. The mouse C-terminal flanking peptide, the GMAP, which is encoded on the same mRNA as galanin, shows a high degree of homology with all other known GMAP sequences but is not identical to any of them and it is more charged than the other GMAP sequences. Synthetic mouse galanin was found to displace [125I]mono-iodo-Tyr26 galanin (porcine) from receptors in the mouse hypothalamic membranes with high affinity (KD = 0.9 nM). Estrogen treatment of mice (0.1 mg/kg i.p.) for 6 h, which elevates the rat pituitary galanin mRNA levels, does not affect the galanin mRNA levels in mouse hypothalamus and pituitary. Neither does a subchronic glucocorticoid treatment (dexamethasone, 0.5 mg/kg i.p. for 7 days) affect these mRNA levels.

Dicationic furans inhibit development of Cryptosporidium parvum in HSD/ICR suckling Swiss mice.

The efficacy of dicationic diarylfurans was evaluated against Cryptosporidium parvum by a suckling murine model. Candidate drugs were solubilized or suspended in deionized water and administered orally at a constant dose rate on days 0-5 (treatment day 0) to suckling ICR Swiss mice experimentally inoculated with oocysts of C. parvum. Efficacy was based on numbers of oocysts recovered from the intestinal tracts of mice subjected to necropsy examination on day 6. Numerous candidate furans significantly reduced the numbers of oocysts recovered from treated mice compared with control mice. Compounds 1, 2, 4, and 9 demonstrated superior efficacies (10% of controls or better) against C. parvum. Compounds 3, 5, 6, 7, 8, 11, 17, 18, and 19 also significantly reduced the numbers of oocysts recovered from treated mice but demonstrated efficacies ranging from 17 to 65% of controls. Compound 4 was particularly efficacious against C. parvum at a dosage as low as 8.5 mg/kg of body weight. Compound 4 is identified as a lead compound for additional studies in other animal models.

What patients think of dental services.

In 1998, research on 'What the general public wants from the general dental service' was carried out by the Centre for Dental Services Studies (CDSS) at the University of York and was commissioned by the British Dental Association (BDA). The research culminated in the report: 'User Priorities for General Dental Services'. This article outlines the main message from the research and contains the researcher's personal observations.

Second-order conditioning of the rabbit's nictitating membrane response. Interstimulus interval and frequency of CS-CS pairings.

Second-order conditioning of the rabbit's nictitating membrane response (NMR) was investigated when second-order trials (CS1-CS2) were intermixed with first-order trials (CS2-US) from the outset of training. Experiment 1 showed that CR acquisition to CS1 was inversely related to the CS1-CS2 interval but nevertheless extended to an interval of 8,400 ms. Experiment 2 revealed that CR acquisition of CS1 was an inverted-U function of the number of CS1-CS2 trials relative to a fixed number of CS2-US trials. Experiment 3 directly contrasted second-order conditioning with a reinforced serial compound procedure (CS1-CS2-US) and a mixed procedure in which second-order trials were intermixed with the reinforced serial compound. Second-order conditioning was about half the strength of either the reinforced serial compound or the mixed procedure, which were similar. The present results are discussed with respect to the relative strength of excitatory and inhibitory processes in second-order conditioning.

Targeting of a human iron-sulfur cluster assembly enzyme, nifs, to different subcellular compartments is regulated through alternative AUG utilization.

Iron-sulfur clusters are prosthetic groups that are required for the function of numerous enzymes in the cell, including enzymes important in respiration, photosynthesis, and nitrogen fixation. Here we report cloning of the human homolog of NifS, a cysteine desulfurase that is proposed to supply the inorganic sulfur in iron-sulfur clusters. In human cells, different forms of NifS that localize either to mitochondria or to the cytosol and nucleus are synthesized from a single transcript through initiation at alternative inframe AUGs, and initiation site selection varies according to the pH of the medium or cytosol. Thus, a novel form of translational regulation permits rapid redistribution of NifS proteins into different compartments of the cell in response to changes in metabolic status.

Design of chimeric peptide ligands to galanin receptors and substance P receptors.

Several chimeric peptides were synthesized and found to be high-affinity ligands for both galanin and substance P receptors in membranes from the rat hypothalamus. The peptide galantide, composed of the N-terminal part of galanin and C-terminal part of substance P (SP), galanin-(1-12)-Pro-SP-(5-11) amide, which is the first galanin antagonist to be reported, recognizes two classes of galanin binding sites (KD(1) less than 0.1 nM and KD(2) approximately 6 nM) in the rat hypothalamus, while it appears to bind to a single population of SP receptors (KD approximately 40 nM). The chimeric peptide has higher affinity towards galanin receptors than the endogenous peptide galanin-(1-29) (KD approximately 1 nM) or its N-terminal fragment galanin-(1-13) (KD approximately 1 microM), which constitutes the N-terminus of the chimeric peptide. Galantide has also higher affinity for the SP receptors than the C-terminal SP fragment-(4-11) amide (KD = 0.4 microM), which constitutes its C-terminal portion. Substitution of amino acid residues, which is of importance for recognition of galanin by galanin receptors, such as [Trp2], in the galanin portion of the chimeric peptide or substitution of ([Phe7] or [Met11]-amide) in the SP portion of chimeric peptide both cause significant loss in affinity of the analogs of galantide for both the galanin- and the SP-receptors. These results suggest that the high affinity of the chimeric peptide, galantide, may in part be accounted for by simultaneous recognition/binding to both receptors.(ABSTRACT TRUNCATED AT 250 WORDS)