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Iron chelation therapy and inhibition of glial nicotinamide adenine dinucleotide phosphate (NADPH) oxidase can both represent possible routes for Alzheimer's disease modifying therapies. The metal hypothesis is largely focused on direct binding of metals to the N-terminal hydrophilic 1-16 domain peptides of Amyloid beta (Aβ) and how they jointly give rise to reactive oxygen species (ROS) production. The cytotoxic effects of Aβ through ROS and metals are mainly studied in neuronal cells using full-length Aβ1-40/42 peptides. Here we study cellularly-derived ROS during 2-60min in response to non-metal associated mid domain Aβ25-35 in microglial Bv2 cells by fluorescence based spectroscopy. We analyze if Aβ25-35 induce ROS production through NADPH oxidase and if the production is sensitive to iron chelation. NADPH oxidase inhibitor diphenyliodonium (DPI) is used to confirm the production of ROS through NADPH oxidase. We modulate cellular iron homeostasis by applying cell permeable iron chelators desferrioxamine (DFO) and deferiprone (DFP). NADPH oxidase subunit gp91-phox level was analyzed by Western blotting. Our results show that Aβ25-35 induces strong ROS production through NADPH oxidase in Bv2 microglial cells. Intracellular iron depletion resulted in restrained Aβ25-35 induced ROS.

Background and objective: Cardiovascular diseases are still a major public health concern in Estonia despite the decline in the mortality rate during the past decade. For better preventive strategies we aimed to investigate the prevalence of cardiovascular disease risk factors and their relations with age, gender and ethnicity.Materials and methods: The cross-sectional study was carried out in Tallinn, Estonia. Two hundred individuals from each of the sex and 10-year age group (range 20–65 years of age) were randomly selected and invited to participate. Final study sample consisted of 511 men and 600 women (mean age of 46 years). Physiological measurements were taken and blood samples were drawn for standard measurements of the following markers: total cholesterol, high- and low-density lipoprotein cholesterol, apolipoproteins, triglycerides, glucose and inflammatory markers.Results: Overall, 31% of the study subjects had high blood pressure, 23% had metabolic syndrome, and 55% were overweight/obese. The prevalence of all risk factors increased with age amongst both genders. The proportion of individuals having increased cholesterol, apolipoprotein B-100, and homocysteine levels was very high amongst both genders (60–80%). More Russians and other ethnic minorities compared to ethnic Estonians had calculated 10-year CHD risk ≥ 10%.Conclusions: The study established a high prevalence of cardiovascular disease risk factors in Estonian adults (20–65 years of age). Younger portion of the population and some extent ethnic considerations should be taken into account when designing future studies, health prevention activities and interventions.

Identifying MAPK pathways and understanding their role in microglial cells may be crucial for understanding the pathogenesis of neurodegenerative diseases since activated microglia could contribute to the progressive nature of neurodegeneration. In this study we show that the JNK pathway plays an important role in the survival of resting microglia BV-2 cells, as evidenced by Annexin-V positive staining and caspase-3 activation in cells treated with the specific JNK inhibitor SP600125. During LPS-induced activation of BV-2 cells inhibition of the p38 and JNK pathways with SB203580 and SP600125, respectively, results in apoptosis as detected by apoptotic markers. In the presence SP600125 the phosphorylation of p38 was significantly increased both in control and LPS-activated BV-2 cells. This suggests that the pro-survival role of JNK is possible due to its abrogation of a potentially apoptotic signal mediated by p38 MAPK pathway. Furthermore, inhibition of the p38 MAPK pathway during LPS-induced activation of BV-2 cells resulted in an increased phosphorylation of c-Jun, suggesting that the pro-survival effect of p38 MAPK during inflammatory conditions involves the JNK pathway. In conclusion, the results of this study demonstrate that both the JNK and p38 MAPK pathways possess anti-apoptotic functions in the microglial cell line BV-2 during LPS-induced activation.

Activated microglia cells, observed during chronic inflammation, produce and secrete compounds that at high concentrations or during sustained production might cause neuronal cell death. Inducible nitric oxide synthase (iNOS) is expressed in response to various immunological stimuli and catalyses the formation of the free radical nitric oxide (NO), that at low and regulated levels participate in cell signaling and cytoprotective events, whereas its higher and unregulated production can promote neurotoxicity in cells or in tissues. Regulation of NO production is therefore central for maintaining NO-levels within a safe window. We have analyzed iNOS protein expression and NO production, in murine microglial Bv-2 cells after 16h treatment with the bacterial endotoxin lipopolysaccharide (LPS). We have further analyzed three MAPK pathways, by co-treating the cells with LPS and the inhibitors of ERK1/2, p38 or JNK MAPK activities. To investigate participation of an oxidative regulatory mechanism, cells were also treated with the antioxidant N-acetyl-L-cysteine (NAC). Our results show that LPS-induced iNOS expression in Bv-2 cells is mainly mediated through JNK MAPK. In addition, co-treatment of the Bv-2 cells with LPS and NAC surprisingly further increased the iNOS expression, an effect also found to be mediated through the JNK MAPK pathway. The level of phosphorylated JNK MAPK (p46) was strongly increased by LPS alone and was further increased when combined with NAC. Our data indicate that iNOS and NO production are suppressed by an oxidative mechanism acting on the JNK MAPK pathway and we speculate that it might constitute a potential regulatory mechanism controlling the NO level.

Neuronal cell line based membrane models were studied as capillary coatings by capillary electrochromatography. The membrane models were based either on membrane suspensions or on membrane lipid extractions. The stability of the coatings at different pH values and buffer compositions was studied. The results showed that the cell membrane suspension based coatings were stable over pH range of 6.5–10.8. The use of Hepes instead of TE buffer did not improve the coating performance.

The molecular mechanism of neurodegeneration in prion diseases remains largely uncertain, but one of the features of infected cells is higher sensitivity to induced oxidative stress. In this study, we have investigated the role of iron in hydrogen peroxide (H(2)O(2))-induced toxicity in scrapie-infected mouse neuroblastoma N2a (ScN 2 a) cells. ScN 2 a cells were significantly more susceptible to H(2)O(2) toxicity than N2a cells as revealed by cell viability (MTT) assay. After 2h exposure, significant decrease in cell viability in ScN 2 a cells was observed at low concentrations of extracellular H(2)O(2) (5-10 microM), whereas N2a cells were not affected. The increased H(2)O(2) toxicity in ScN 2 a cells may be related to intracellular iron status since ferrous iron (Fe(2+)) chelator 2,2'-bipyridyl (BIP) prevented H(2)O(2)-induced decrease in cell viability. Further, the level of calcein-sensitive labile iron pool (LIP) was significantly increased in ScN 2 a cells after H(2)O(2) treatment. Finally, the production of reactive oxygen species (ROS) was inhibited by 30% by iron chelators desferrioxamine (DFO) and BIP in ScN 2 a cells, whereas no significant effect of iron chelators on basal ROS production was observed in N2a cells. This study indicates that cellular resistance to oxidative stress in ScN 2 a cells is associated with intracellular status of reactive iron.

Prion diseases are characterized by the conversion of the normal cellular prion protein PrP(C) into a pathogenic isoform, PrP(Sc). The mechanisms involved in neuronal cell death in prion diseases are largely unknown, but accumulating evidence has demonstrated oxidative impairment along with metal imbalances in scrapie-infected brains. In this study, we report changes in cellular iron metabolism in scrapie-infected mouse neuroblastoma N2a cells (ScN2a). We detected twofold lower total cellular iron and calcein-chelatable cytosolic labile iron pool (LIP) in ScN2a cells as compared to the N2a cells. We also measured in ScN2a cells significantly lower activities of iron regulatory proteins 1 and 2 (IRP1 and IRP2, respectively), regulators of cellular iron by sensing cytosolic free iron levels and controlling posttranscriptionally the expression of the major iron transport protein transferrin receptor 1 (TfR1) and the iron sequestration protein ferritin. IRP1 and IRP2 protein levels were decreased by 40% and 50%, respectively, in ScN2a cells. TfR1 protein levels were fourfold reduced and ferritin levels were threefold reduced in ScN2a cells. TfR1 and ferritin mRNA levels were significantly reduced in ScN2a cells. ScN2a cells responded normally to iron and iron chelator treatment with respect to the activities of IRP1 and IRP2, and biosynthesis of TfR1 and ferritin. However, the activities of IRP1 and IRP2, and protein levels of TfR1 and ferritin, were still significantly lower in iron-depleted ScN2a cells as compared to the N2a cells, suggesting lower need for iron in ScN2a cells. Our results demonstrate that scrapie infection leads to changes in cellular iron metabolism, affecting both total cellular and cytosolic free iron, and the activities and expression of major regulators of cellular iron homeostasis.

We report the effects of amyloid precursor protein (APP) fragment 714-723 (APP(714-723); peptide P1) and its V717F and V717G mutants (peptides P2 and P3, respectively) on G-protein activity ([35S]GTPgammaS binding) in membranes from postmortem human control and Alzheimer's disease (AD) brains. The peptides P1, P2, and P3 revealed a significant stimulatory effect on [35S]GTPgammaS binding in control temporal cortex. The most potent stimulator, P3, at 10 microM concentration enhanced [35S]GTPgammaS binding by 500%. The effect was threefold stronger than that for wild-type P1 and twofold stronger than that for P2. In sporadic AD, the stimulatory effect of P1, P2, and P3 on G-proteins was reduced significantly whereas in Swedish familial AD (SFAD), only P1 elicited marked stimulation (at 10 microM by 50%). In control sensory postcentral cortex, the stimulation of G-proteins by P3 was 1.5-fold lower than that in control temporal cortex, whereas in AD and SFAD the effect showed no remarkable regional difference. Treatment of membranes with H2O2 produced 1.5-fold higher stimulation in [35S]GTPgammaS binding to temporal cortex than that in binding to sensory postcentral cortex. In AD and SFAD, the stimulation by H2O2 revealed no significant regional difference. Glutathione, desferrioxamine (DFO), and 17beta-estradiol markedly decreased the strong stimulatory effect by P3 on [35S]GTPgammaS binding to control temporal cortex, with the protective effect by DFO being most potent. The G(alphaO)-protein levels were not changed in AD or SFAD brain membranes as compared to levels in control membranes. We suggest that strong G-protein stimulation by P3 in the human brain implies the specific (per)oxidation mechanism that might be affected by regional content of peroxidizing substrates and antioxidants.

In this work, we have targeted positions 18-38 of the human galanin receptor type 1 (GalR1) mRNA coding sequence with different peptide nucleic acid (PNA) oligomers. This region has previously been shown to be a good antisense region and therefore we aimed to identify the subregions and/or thermodynamic parameters determining the antisense efficacy. Nine different PNA oligomers were conjugated to a cell-penetrating peptide, transportan, to enhance their cellular uptake. Concentration-dependent down-regulation of GalR1 protein expression in human melanoma cell line Bowes was measured by radioligand binding assay. No reduction of GalR1 mRNA level was observed upon PNA treatment, thus, the effect was concluded to be translational arrest. Judging from the EC50 values, antisense PNA oligomers targeting regions 24-38 (EC50=70 nM) or 27-38 (EC50=80 nM) were the most potent suppressors of protein expression. No parameter predicted by M-fold algorithm was found to correlate with the measured antisense activities. Presence of some subregions was found not to increase antisense efficiency of PNA. Presence of a short unpaired triplet between nucleotides 33 and 35 in the target region was, on the other hand, found to be the most critical for efficient GalR1 down-regulation. Thus, the results are of high impact in designing antisense oligomers. Specific results of this study demonstrate 20-fold more efficient antisense down-regulation of GalR1 as achieved before.

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.

The galanin-receptor ligand M40 [galanin-(1-12)-Pro3-(Ala-Leu)2-Ala amide] binds with high affinity to [mono[125I]iodo-Tyr26]galanin-binding sites in hippocampal, hypothalamic, and spinal cord membranes and in membranes from Rin m5F rat insulinoma cells (IC50 = 3-15 nM). Receptor autoradiographic studies show that M40 (1 microM) displaces [mono[125I]iodo-Tyr26]galanin from binding sites in the hippocampus, hypothalamus, and spinal cord. In the brain, M40 acts as a potent galanin-receptor antagonist: M40, in doses comparable to that of galanin, antagonizes the stimulatory effects of galanin on feeding, and it blocks the galaninergic inhibition of the scopolamine-induced acetylcholine release in the ventral hippocampus in vivo. In contrast, M40 completely fails to antagonize both the galanin-mediated inhibition of the glucose-induced insulin release in isolated mouse pancreatic islets and the inhibitory effects of galanin on the forskolin-stimulated accumulation of 3',5'-cAMP in Rin m5F cells; instead M40 is a weak agonist at the galanin receptors in these two systems. M40 acts as a weak antagonist of galanin in the spinal flexor reflex model. These results suggest that at least two subtypes of the galanin receptor may exist. Hypothalamic and hippocampal galanin receptors represent a putative central galanin-receptor subtype (GL-1-receptor) that is blocked by M40. The pancreatic galanin receptor may represent another subtype (GL-2-receptor) that recognizes M40, but as a weak agonist. The galanin receptors in the spinal cord occupy an intermediate position between these two putative subtypes.

The 25 amino acid residue chimeric peptide M32, galanin(1-13)-neuropeptide Y(25-36)-amide, was synthesized. The peptide was found to be recognized by both galanin and NPY receptors. The solution structure in 30% (v/v) 1,1,1,3,3,3-hexafluoro-2-propanol was examined by 2-D 1H-NMR and by CD. Proton resonance assignments were made, and structures were calculated using DIANA and refined by restrained energy minimization and molecular dynamics. The obtained structures contain an alpha-helical part in the NPY portion of the peptide including residues 13-20, and in some structures it continues to the C-terminal Tyr25. The more flexible N-terminal portion of the peptide has the freedom to approach the C-terminal alpha-helix, via a reverse turn or a nascent alpha-helix, which permits the N-terminus with Trp2 to come into close contact with the C-terminus with Tyr25. Among the ten NMR structures with lowest energy, there are structures reminiscent of the horseshoe shape of aPP, a close relative of NPY with known crystal structure. It appears that the strong alpha-helical character of the NPY (25-36) amide fragment of M32 helps to stabilize structural features in the galanin-derived part of the peptide. It is noteworthy that this rigid NPY portion of M32 does not prevent the recognition of the peptide by galanin receptors; rather, the peptide has unusually high affinity: IC50 = 0.1 nM at galanin receptors. The chimeric peptide M32 is also recognized by NPY receptors with submicromolar affinity (IC50 = 0.25 microM). The availability of a solution structure for peptide M32, which is recognized by two peptide receptors that are both members of the family of G-protein-coupled receptors, may be useful in understanding peptide receptor-ligand interactions and in designing new galanin and NPY receptor ligands.

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.

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.

Novel stationary phases in open tubular CEC were investigated. The coating procedure was fast and simple. The coating material contained membrane suspension of different neuronal cell lines. The performance and stability of three cell lines: human neuroblastoma SH-SY5Y, murine microglia Bv-2 and human glioma U87-MG cells were studied. The coating solution was expected to contain both membrane proteins and membrane lipids. The presence of membrane proteins was tested by Western blotting and the presence of phospholipids by the analysis of phosphorus content. The stability of the coating was estimated by monitoring the mobility of EOF over successive runs. The effects of pH, storage time and temperature on the coating stability were also studied. The results showed that the cell membrane-based coating was stable over pH range of 6.5-8.5. Coatings derived from different cells yielded similar stability and EOF mobility. Capillary coated with a membrane solution was stable over 3-day period. The same coating solution could be used for 3 weeks.

Preface Further Reading Biography Introduction to Part 1 Matjaz Zorko From Building Block to 3-D Structure Amino Acids Matjaz Zorko Noncovalent Interactions Matjaz Zorko Structural Organization of Proteins Matjaz Zorko Protein Biosynthesis and Posttranslational Modifications The Biosynthesis of Proteins Matjaz Zorko Posttranslational Modifications Matjaz Zorko Folding of Proteins Protein Folding Matjaz Zorko Intracellular Sorting of Proteins Gunnar von Heijne Protein Degradation Protein Turnover Matjaz Zorko Introduction to Part II Astrid Graslund Purification and Characterization of Proteins Matjaz Zorko Crystallography and X-Ray Diffraction Astrid Graslund Optical Spectroscopy Astrid Graslund Nuclear Magnetic Resonance (NMR) Astrid Graslund Methods to Follow Protein Folding Astrid Graslund Mass Spectrometry Astrid Graslund Chemical Synthesis of Peptides and Proteins Ulo Langel Introduction to Part III Tiit Land Protein Engineering and Gene Silencing Ulo Langel Protein-Ligand Interactions Ulo Langel Sequence Analysis and Function Prediction Tiit Land Protein Structure Prediction Tiit Land Proteomics Sherry Niessen and Benjamin F. Cravatt Introduction to Part IV Ulo Langel Enzymes Matjaz Zorko Nucleic Acid-Binding Proteins Ulo Langel Cell Surface Receptors and Signaling Ulo Langel Membrane Proteins Gunnar von Heijne Antibodies Ulo Langel Fibrous Proteins Ulo Langel Selected Classes of Bioactive Peptides Ulo Langel Introduction to Part V Ulo Langel and Astrid Graslund Misfolding-Based Diseases Astrid Graslund Miscleavage-Based Diseases Astrid Graslund Missequence-Based Diseases Astrid Graslund Peptides and Proteins as Drugs Ulo Langel

The 20-amino acid peptide M-15 binds with high affinity (IC50 approximately 0.1 nM) to 125I-labeled galanin (125I-GAL) binding sites in membranes from the ventral hippocampus, midbrain, and rat spinal cord. Receptor autoradiographic studies show that M-15 can displace 125I-GAL from all labeled sites. M-15 acts as a reversible high-affinity antagonist in blocking the inhibitory effects of GAL on the evoked release of acetylcholine in vivo in the hippocampus and on the GAL-induced hyperpolarization of locus coeruleus neurons in slices. M-15 also blocks the facilitatory effects of GAL on the spinal flexor reflex. Thus, the chimeric peptide M-15 [GAL-(1-13)-substance P-(5-11)amide] represents the first antagonist to the neuronal actions of GAL.

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).

The activation of cellular inflammatory response is tightly linked to induced production of reactive oxygen species (ROS) and nitric oxide (NO), which in turn have been identified as important regulators of cellular iron metabolism. In the present study, we have used the microglia cell line BV-2 and the neuroblastoma cell line N2a to study the regulatory effects of the microbial agent lipopolysaccharide (LPS) on the expression of the transferrin receptor (TfR) and ferritin in cell lines with different characteristics. The receptor mainly responsible for LPS recognition is the Toll-like receptor 4 (TLR4) that triggers a variety of intracellular signalling cascades leading to the induction of transcription of target genes involved in the innate immune response. Among the pathways to be activated is the MAPK cascade leading to the activation of nuclear factor-kappaB that induces transcription of a variety of genes, e.g., inducible nitric oxide synthase (iNOS). The TLR4-mediated LPS response also induces the production of ROS through a mechanism(s) suggested to involve the activation of NADPH oxidase(s). This study shows that exposure of BV-2 and N2a cells to LPS results in decreased TfR protein levels and increased H-ferritin mRNA levels. The LPS down-regulatory effect on TfR protein expression is abolished by the NADPH oxidase inhibitor diphenyliodonium (DPI) but is not affected by the free radical scavenger N-acetyl-L-cysteine (NAC) or the iNOS inhibitor aminoguanidine (AG). The increased H-ferritin mRNA levels in response to LPS are not affected by DPI, NAC, or AG.

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.

A linkage between sulfur and iron metabolism has been suggested since sulfide has the ability to release iron from ferritin in the presence of iron acceptors in vitro. Nevertheless, this linkage is still lacking evidence in vivo as well as in cellular models. In this study we have treated human RD4 skeletal muscle cells with sodium sulfide and measured the level of the labile iron pool (LIP) as well as the intracellular sulfide concentration. We have also detected the amounts of L-ferritin protein as well as the iron regulatory protein 2 (IRP2). The sulfide treatment resulted in a 100% increase in the amount of LIP after 1 and 2 h. We also found that the raise of the LIP levels was coupled to an elevation of the amounts of intracellular sulfide that increased by 60%. The bioavailability of the released iron was confirmed by a 100% increase in L-ferritin protein as well as a 60% decrease of the IRP2 protein levels. These results suggest that there is a linkage between sulfur metabolism and intracellular iron regulation in mammalian cells.

The influence of three C-terminal sequences and of transmembrane domain from amyloid precursor protein (APP) on the activity of G-proteins and of the coupled cAMP-signalling system in the postmortem Alzheimer’s disease (AD) and age-matched control brains was compared. 10 μM APP(639–648)–APP(657–676) (PEP1) causes a fivefold stimulation in the [ 35 S]GTPγS-binding to control hippocampal G-proteins. APP(657–676) (PEP2) and APP(639–648) (PEP4) showed less pronounced stimulation whereas cytosolic APP(649–669) (PEP3) showed no regulatory activity in the [ 35 S]GTPγS-binding. PEP1 also showed 1.4-fold stimulatory effect of on the high-affinity GTPase and adenylate cyclase activity in control membranes, whereas in AD hippocampal membranes the stimulatory effect of PEP1 was substantially weaker. The PEP1 stimulation of the [ 35 S]GTPγS-binding to the control membranes was significantly reduced by 1.5 mM glutathione, 0.5 mM antioxidant N -acetylcysteine and, in the greatest extent, by 0.01 mM of desferrioxamine. In AD hippocampus these antioxidants revealed no remarkable reducing effect on PEP1-induced stimulation. Our results suggest that C-terminal and transmembrane APP sequences possess receptor-like G-protein activating function in human hippocampus and that abnormalities of this function contribute to AD progression. The stimulatory action of these sequences on G-protein mediated signalling suggests the region-specific formation of reactive species.

Galanin is a neuropeptide initiating its multiple biological effects by binding to different subtypes of seven transmembrane spanning galanin receptors, thus transducing the extracellular signal to G-proteins. There is a wide range of galanin receptor ligands available today, and some of the ligands have been shown to act as antagonists in galaninergic signal transduction systems at tissue level and in vivo. Subtype-specific galanin receptor ligands could be used selectively to initiate or block the effects of galanin in order to provide insights into the role of galanin both at normal and pathophysiological conditions, and in the development of compounds with therapeutical values. This review highlights the recent reports on the binding affinities of GalR in CNS and transfected cell lines.

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 in-frame 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.

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.

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=0.9nM). 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.

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)

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.

Publisher Summary This chapter discusses the methods for radiolabeling galanin with 125 I. Kinetic studies are carried out under pseudo first-order kinetic conditions using 125 I-labeled galanin in at least 10-fold excess over the receptor concentration. In binding studies with neuropeptides, one has to determine whether the binding versus time curve shows a maximum followed by decline or whether saturation is achieved when incubation time increases. N-terminal fragment, which is fully conserved with respect to amino acid sequence among rat, porcine, and bovine galanin, is a high-affinity agonist at pancreatic and at hippocampal galanin receptors. The only reported cases where C-terminal galanin fragments are active alone or together with N-terminal ones concern the gastric smooth muscle in rat.

Galanin is a neuroendocrine peptide which is 29/30 amino acids in length and is recognised by G-protein-coupled central nervous system receptors via its N-terminus. We synthesised several galanin receptor ligands and fragments around C-terminal extensions of galanin(1-13) to yield chimeric peptides with C-terminals corresponding to bioactive peptides like bradykinin(2-9), mastoparan, neuropeptide Y(25-36) or substance P(5-11), respectively. We also synthesised short galanin analogs in which galanin(1-13) was C-terminally elongated with Lys14; different pharmacologically active small molecules were then attached to the epsilon-amino group of Lys14. Several cysteine-substituted linear and ring closed analogs of galanin(1-9) and galanin(1-16) were also synthesised. The equilibrium binding constants for these peptides at hypothalamic galanin receptors were determined and found in the subnanomolar to micromolar range. The large number of peptides and their binding affinities presented here permit structure-activity relationship analysis of peptide-type ligands to galanin receptors.