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Immunoprecipitation of mRNA-protein complexes is a method that can be used to study RNA binding protein (RBP)-RNA interactions. In this protocol, an antibody targeting an RBP of interest is used to immunoprecipitate the RBP and any interacting molecules from a cell lysate. Reverse transcription followed by PCR is then used to identify individual mRNAs isolated with the RBP. This method focuses on examining an association between a specific RBP-mRNA complex, and it is best suited for a small scale screening of known or putative binding partners. It can also be used as a second, independent method to verify RBP-mRNA interactions discovered through more universal screening techniques. We describe the immunoprecipitation protocol in practical detail and discuss variations of the method as well as issues associated with it. The procedure takes three days to complete.

Galanin is a 29/30 amino acid peptide neurotransmitter that is widely distributed throughout the central nervous system and periphery. There are three well-characterized G-protein coupled galanin receptors subtypes (GalR1-3). A more recently discovered 60 amino acid galanin-like peptide (GALP) shares amino acid sequence homology with galanin (1-13) in position 9-21 and has high binding affinity for GalR1-3, with highest affinity for GalR3. Considerable evidence has accumulated that implicates both galanin and GALP as playing important roles in regulating food and water intake behavior and related neuroendocrine functions. Pharmacological tools are emerging that will allow dissociation of specific roles for the peptides and their associated receptor subtypes in mediating the homeostatic processes of energy and fluid balance.

The search for antiepileptic drugs that are capable of blocking the progression of epilepsy (epileptogenesis) is an important problem of translational epilepsy research. The neuropeptide galanin effectively suppresses acute seizures. We examined the ability of hippocampal galanin receptor type 1 (GalR1) and type 2 (GalR2) to inhibit kindling epileptogenesis and studied signaling cascades that mediate their effects. Wistar rats received 24-h-long intrahippocampal infusion of a GalR1/2 agonist galanin(1-29), GalR1 agonist M617 [galanin(1-13)-Gln14-bradykinin(2-9)-amide], or GalR2 agonist galanin(2-11). The peptides were administered alone or combined with an inhibitor of Gi protein pertussis toxin (PTX), Gi-protein activated K+ channels (GIRK) inhibitor tertiapin Q (TPQ), G(q/11) protein inhibitor [D-Arg1,D-Trp(5,7,9),Leu11]-substance P (dSP), or an inhibitor of intracellular Ca2+ release dantrolene. Sixteen hours into drug delivery, the animals were subjected to rapid kindling-60 electrical trains administered to ventral hippocampus every 5 min. M617 delayed epileptogenesis, whereas galanin(1-29) and galanin(2-11) completely prevented the occurrence of full kindled seizures. TPQ abolished anticonvulsant effect of M617 but not of galanin(2-11). PTX blocked anticonvulsant effects of M617 and inversed the action of galanin(1-29) and galanin(2-11) to proconvulsant. dSP and dantrolene did not modify seizure suppression through GalR1 and GalR2, but eliminated the proconvulsant effect of PTX + galanin(1-29) and PTX + galanin(2-11) combinations. We conclude that hippocampal GalR1 exert their disease-modifying effect through the Gi-GIRK pathway. GalR2 is antiepileptogenic through the Gi mechanism independent of GIRK. A secondary proconvulsant pathway coupled to GalR2 involves G(q/11) and intracellular Ca2+. The data are important for understanding endogenous mechanisms regulating epileptogenesis and for the development of novel antiepileptogenic drugs.

Basic peptides with vector abilities, so called cell-penetrating peptides (CPPs), have been reported to enter cells, carrying cargoes ranging from oligonucleotides and proteins to nanoparticles. In this study we present novel CPPs derived from the gamma-secretase complex, which is involved in the amyloidogenic processing of the amyloid precursor protein (APP) and one of the major research targets for Alzheimer’s disease therapeutics today. In order to examine the uptake efficiency and internalization mechanism of these novel CPPs, side-by-side comparison with the well characterized CPPs penetratin and tat were made. For assessment of the CPP uptake mechanism, endocytosis inhibitors, endosomal markers and cells deficient in the expression of glycosaminoglycans were used. Also, in order to determine the vector ability of the peptides, protein delivery was quantified. We demonstrate the uptake of the gamma-secretase derived CPPs, in accordance to penetratin and tat, to be largely dependent on temperature and initial binding to cell-surface glycosaminoglycans. After this initial step, there is a discrepancy in the mechanism of uptake, where all peptides, except one, is taken up by a PI 3-kinase dependent fluid phase endocytosis, which could be inhibited by wortmannin. Also, by using endosomal markers and protein delivery efficacy, we conclude that the pathway of internalization for different CPPs could determine the possible cargo size for which they can be used as a vector. The, in this study demonstrated, cell-penetrating properties of the gamma-secretase constituents could prove to be of importance for the gamma-secretase function, which is a matter of further investigation.

Galanin and its three receptors have been linked to a wide variety of physiological processes and are distributed in both the central and peripheral nervous systems. Further knowledge of the proper ...

To understand the role of RNA-binding proteins (RBPs) in the regulation of gene expression, methods are needed for the in vivo identification of RNA-protein interactions. We have developed the peptide nucleic acid (PNA)-assisted identification of RBP technology to enable the identification of proteins that complex with a target RNA in vivo . Specific regions of the 3′ and 5′ UTRs of ankylosis mRNA were targeted by antisense PNAs transported into cortical neurons by the cell-penetrating peptide transportan 10. An array of proteins was isolated in complex with or near the targeted regions of the ankylosis mRNA through UV-induced crosslinking of the annealed PNA-RNA-RBP complex. The first evidence for pharmacological modulation of these specific protein-RNA associations was observed. These data show that the PNA-assisted identification of the RBP technique is a reliable method to rapidly identify proteins interacting in vivo with the target RNA.

Previous studies identified the effector protein YopM of Yersinia enterocolitica as a novel bacterial cell-penetrating protein. YopM’s ability to translocate across the host cell plasma membrane independently of Yersinia’s type III secretion system (T3SS) is mediated by its two N-terminal α-helices. The SspH1 effector protein of Salmonella typhimurium shares significant homology in sequence and structure with YopM, which prompted us to investigate potential cell-penetrating abilities of this effector protein. For this, we recombinantly expressed SspH1 in Escherichia coli and analyzed a potential T3SS-independent translocation of the protein by cell fractionation of HeLa cells, immunofluorescence microscopy and FACS analyses. The functionality of the recombinant protein as an E3 ubiquitin- ligase was determined using in vitro -ubiquitination assays. Additionally, an effect of the recombinant protein on the expression of pro-inflammatory cytokines was analyzed by quantitative real time PCR. In this study, we could show that the SspH1 effector protein of Salmonella typhimurium is able to translocate autonomously into eukaryotic cells without requiring additional factors. Furthermore, we could show that recombinant SspH1 is a functional E3 ubiquitin ligase that is able to undergo auto-ubiquitination following T3SS-independent translocation and to reduce the expression of Interleukin-8 in IL-1β stimulated cells.

Cell-penetrating peptides have proven themselves as valuable vectors for intracellular delivery. Relatively little is known about the frequency of cell-penetrating sequences in native proteins and their functional role. By computational comparison of peptide sequences, we recently predicted that intracellular loops of G-protein coupled receptors (GPCR) have high probability for occurrence of cell-penetrating motifs. Since the loops are also receptor and G-protein interaction sites, we postulated that the short cell-penetrating peptides, derived from GPCR, when applied extracellularly can pass the membrane and modulate G-protein activity similarly to parent receptor proteins. Two model systems were analyzed as proofs of the principle. A peptide based on the C-terminal intracellular sequence of the rat angiotensin receptor (AT1AR) is shown to internalize into live cells and elicit blood vessel contraction even in the presence of AT1AR antagonist Sar1-Thr8-angiotensin II. The peptide interacts with the same selectivity towards G-protein subtypes as agonist-activated AT1AR and blockade of phospholipase C abolishes its effect. Another cell-penetrating peptide, G53-2 derived from human glucagon-like peptide receptor (GLP-1R) is shown to induce insulin release from isolated pancreatic islets. The mechanism was again found to be shared with the original GLP-1R, namely G11-mediated inositol 1,4,5-triphosphate release pathway. These data reveal a novel possibility to mimic the effects of signalling transmembrane proteins by application of shorter peptide fragments.

Galnon was first reported as a low molecular weight non-peptide agonist at galanin receptors [Saar et al. (2002) Proc. Natl. Acad. Sci. USA 99 , 7136–7141]. Following its systemic administration, this synthetic ligand affected a range of important physiological processes including appetite, seizures and pain. Physiological activity of galnon could not be explained solely by the activation of the three known galanin receptors, GalR1, GalR2 and GalR3. Consequently, it was possible that galnon generates its manifold effects by interacting with other signaling pathway components, in addition to via GalR1-3. In this report, we establish that galnon: (i) can penetrate across the plasma membrane of cells, (ii) can activate intracellular G-proteins directly independent of receptor activation thereby triggering downstream signaling, (iii) demonstrates selectivity for different G-proteins, and (iiii) is a ligand to other G-protein coupled receptors (GPCRs) in addition to via GalR1-3. We conclude that galnon has multiple sites of interaction within the GPCR signaling cascade which mediate its physiological effects.

A major obstacle in the development of new therapeutic agents is the low bioavailability of hydrophilic substances. Drugs that bind to intracellular targets must penetrate the lipid bilayer surrounding the cell in order to exert their effect. A relatively new research area that addresses this problem by introducing novel transport peptides that facilitate the cellular penetration of potential drugs has emerged. These peptides predominantly have a positive net charge and/or an amphipathic nature, but can otherwise have very different characteristics. This group of peptides, although sometimes called protein transduction domains (PTDs), is here referred to as cell-penetrating peptides (CPPs). For many years it was believed that these peptides were internalized into cells via a non-endocytotic, receptor-independent pathway. However, recent publications have suggested that an endocytotic pathway cannot be ruled out, and that earlier results might be based on artifacts associated with fixation of cells and membrane association of peptides. Although the mechanism of cellular uptake remains unclear, there is an increasing amount of reports on biological effects of CPPs and their cargos. Thus, CPPs are an attractive pharmaceutical and biochemical tool that needs more attention. This review will discuss some recent results in this research field with focus on the cell-penetrating peptide transportan.

Attempts to unravel the cell translocation mechanism of a growing number of cell-penetrating peptides (CPP) have revealed molecular determinants essential for internalization ability. The peptide sequence and the charge have been proposed to be the major factors in determining the membrane interaction mode and subsequent internalization pathway. Recent research in this field has shifted to search and design of novel CPPs with predefined vectorial properties and elucidation of the mechanism of cell entry of CPPs with high cargo delivery efficiency. Here we present a map of interaction modes with cell surface and intracellular traffic of transportan and its analogue TP10 complexed with fluorescently labeled avidin or streptavidin-gold conjugates. The protein cargo complexed with either peptide is transduced into HeLa and Bowes cells mostly in the endocytic vesicles with heterogeneous morphology and size as demonstrated by transmission electron microscopy (TEM) and confocal laser scanning fluorescence microscopy. Most of the induced vesicles are large, with 0.5-2 mum diameter, probably macropinosomes, but the complexes are present also in smaller vesicles, suggesting involvement of different pathways. Later the majority of complexes are translocated from the cell periphery into vesicles of perinuclear region and partly to lysosomes. A fraction of transportan-streptavidin complexes is present also freely in cytoplasm, both in the close vicinity of plasma membrane and more centrally, suggesting the escape from endosomal vesicles, since vesicles with discontinuous membrane were also detected by TEM. The cell-translocation process of transportan-protein complexes is temperature dependent and strongly inhibited at 8-10 degrees C and blocked at 4 degrees C when only interaction with the plasma membrane takes place.

Cell-penetrating peptides (CPPs) constitute a new class of delivery vectors with high pharmaceutical potential. However, the abilities of these peptides to translocate through cell membranes can be accompanied by toxic effects resulting from membrane perturbation at higher peptide concentrations. Therefore, we investigated membrane toxicity of five peptides with well-documented cell-penetrating properties, pAntp(43-58), pTAT(48-60), pVEC(615-632), model amphipathic peptide (MAP), and transportan 10, on two human cancer cell lines, K562 (erythroleukemia) and MDA-MB-231 (breast cancer), as well as on immortalized aortic endothelial cells. We studied the effects of these five peptides on the leakage of lactate dehydrogenase and on the fluorescence of plasma membrane potentiometric dye bis-oxonol. In all cell lines, pAntp(43-58), pTAT(48-60), and pVEC(615-632) induced either no leakage or low leakage of lactate dehydrogenase, accompanied by modest changes in bis-oxonol fluorescence. MAP and transportan 10 caused significant leakage; in K562 and MDA-MB-231 cells, 40% of total lactate dehydrogenase leaked out during 10 min exposure to 10 microM of transportan 10 and MAP, accompanied by a significant increase in bis-oxonol fluorescence. However, none of the CPPs tested had a hemolytic effect on bovine erythrocytes comparable to mastoparan 7. The toxicity profiles presented in the current study are of importance when selecting CPPs for different applications.

Alternative splicing of mouse beta3-adrenoceptor transcripts produces an additional receptor isoform (beta3b-adrenoceptor) with a C terminus comprising 17 amino acids distinct from the 13 in the known receptor (beta3a-adrenoceptor). We have shown that the beta3b-adrenoceptor couples to both Gs and Gi, whereas the beta3a-adrenoceptor couples only to Gs. To define the regions involved in this differential G protein coupling, we have compared wild-type, truncated, and mutant beta3-adrenoceptors. In Chinese hamster ovary cells expressing beta3-adrenoceptors truncated at the splicing point, cAMP accumulation with CL316243 [(R,R)-5-[2-[[2-(3-chlorophenyl)-2-hydroxyethyl]-amino]-propyl]1,3-benzodioxole-2,2-dicarboxylate] increased by 59% following pretreatment with pertussis toxin, suggesting that the C-terminal region of the beta3a-adrenoceptor inhibits coupling to Gi. We next utilized the cell-penetrating peptide Transportan 10 (Tp10) to introduce peptides comprising the different C-terminal tail fragments into cells expressing beta3a-adrenoceptor, beta3b-adrenoceptor, and the truncated beta3-adrenoceptor. Treatment with beta3a-Tp10 (1 microM) caused cAMP responses to CL316243 in the beta3a-adrenoceptor to become pertussis toxin-sensitive and display a 30% increase over control, whereas the other peptides did not affect any receptor. Mutation at a potential tyrosine phosphorylation site (Tyr392Ala beta3a-adrenoceptor) did not alter responses or pertussis toxin sensitivity relative to the parent receptor. Surprisingly, a Ser388Ala/Ser389Ala mutant beta3b-adrenoceptor became unresponsive to CL316243 while retaining an extracellular acidification rate response to SR59230A [3-(2-ethylphenoxy)-1-[(1,S)-1,2,3,4-tetrahydronapth-1-ylamino]-2S-2-propanol oxalate]. Our findings suggest that the beta3a-adrenoceptor cannot couple to Gi because of conformational changes induced by a protein(s) that interacts with residues in the C-terminal tail or because this protein(s) affects the intracellular localization of the beta3a-adrenoceptor.

The uptake of different cell-penetrating peptides (CPPs) in two yeast species, Saccharomyces cerevisiae and Candida albicans, was studied using fluorescence HPLC-analyses of cell content. Comparison of the ability of penetratin, pVEC and (KFF)3K to traverse the yeast cell envelope shows that the cellular uptake of the peptides varies widely. Moreover, the intracellular degradation of the CPPs studied varies from complete stability to complete degradation. We show that intracellular degradation into membrane impermeable products can significantly contribute to the fluorescence signal. pVEC displayed highest internalizing capacity, and considering its stability in both yeast species, it is an attractive candidate for further studies.

Several peptides, including penetratin and Tat, are known to translocate across the plasma membrane. Dynorphin opioid peptides are similar to cell-penetrating peptides in a high content of basic and hydrophobic amino acid residues. We demonstrate that dynorphin A and big dynorphin, consisting of dynorphins A and B, can penetrate into neurons and non-neuronal cells using confocal fluorescence microscopy/immunolabeling. The peptide distribution was characterized by cytoplasmic labeling with minimal signal in the cell nucleus and on the plasma membrane. Translocated peptides were associated with the endoplasmic reticulum but not with the Golgi apparatus or clathrin-coated endocytotic vesicles. Rapid entry of dynorphin A into the cytoplasm of live cells was revealed by fluorescence correlation spectroscopy. The translocation potential of dynorphin A was comparable with that of transportan-10, a prototypical cell-penetrating peptide. A central big dynorphin fragment, which retains all basic amino acids, and dynorphin B did not enter the cells. The latter two peptides interacted with negatively charged phospholipid vesicles similarly to big dynorphin and dynorphin A, suggesting that interactions of these peptides with phospholipids in the plasma membrane are not impaired. Translocation was not mediated via opioid receptors. The potential of dynorphins to penetrate into cells correlates with their ability to induce non-opioid effects in animals. Translocation across the plasma membrane may represent a previously unknown mechanism by which dynorphins can signal information to the cell interior.

There is considerable evidence that galanin plays an important role in food intake, nociception, stress reactivity, sexual behavior, and learning. However, its involvement in other important behavioral processes, especially higher cognition, has not been systematically investigated. This review summarizes our recent efforts to explore the role of galanin in executive processes as well as in fundamental motivational and motor productive processes that are essential to the behavioral assessment of cognitive constructs in rodents. Our work points to a role in reward processes and physiological water balance regulation, and argues against a role for galanin in fine motor production and maintenance, simple visual discrimination, and perseveration.

Two novel metalloproteases from Arabidopsis thaliana, termed AtPrePI and AtPrePII, were recently identified and shown to degrade targeting peptides in mitochondria and chloroplasts using an ambiguous targeting peptide. AtPrePI and AtPrePII are classified as dually targeted proteins as they are targeted to both mitochondria and chloroplasts. Both proteases harbour an inverted metal binding motif and belong to the pitrilysin subfamily A. Here we have investigated the subsite specificity of AtPrePI and AtPrePII by studying their proteolytic activity against the mitochondrial F(1)beta pre-sequence, peptides derived from the F(1)beta pre-sequence as well as non-mitochondrial peptides and proteins. The degradation products were analysed, identified by MALDI-TOF spectrometry and superimposed on the 3D structure of the F(1)beta pre-sequence. AtPrePI and AtPrePII cleaved peptides that are in the range of 10 to 65 amino acid residues, whereas folded or longer unfolded peptides and small proteins were not degraded. Both proteases showed preference for basic amino acids in the P(1) position and small, uncharged amino acids or serine residues in the P'(1) position. Interestingly, both AtPrePI and AtPrePII cleaved almost exclusively towards the ends of the alpha-helical elements of the F(1)beta pre-sequence. However, AtPrePI showed a preference for the N-terminal amphiphilic alpha-helix and positively charged amino acid residues and degraded the F(1)beta pre-sequence into 10-16 amino acid fragments, whereas AtPrePII did not show any positional preference and degraded the F(1)beta pre-sequence into 10-23 amino acid fragments. In conclusion, despite the high sequence identity between AtPrePI and AtPrePII and similarities in cleavage specificities, cleavage site recognition differs for both proteases and is context and structure dependent.

Cells are protected from the surrounding environment by plasma membrane which is impenetrable for most hydrophilic molecules. In the last 10 years cell-penetrating peptides (CPPs) have been discovered and developed. CPPs enter mammalian cells and carry cargo molecules over the plasma membrane with a molecular weight several times their own. Known transformation methods for plant cells have relatively low efficiency and require improvement. The possibility to use CPPs as potential delivery vectors for internalisation in plant cells has been studied in the present work. We analyse and compare the uptake of the fluorescein-labeled CPPs, transportan, TP10, penetratin and pVEC in Bowes human melanoma cells and Nicotiana tabacum cultivar (cv.) SR-1 protoplasts (plant cells without cell wall). We study the internalisation efficiency of CPPs with fluorescence microscopy, spectrofluorometry and fluorescence-activated cell sorter (FACS). All methods indicate, for the first time, that these CPPs can internalise into N. tabacum cv. SR-1 protoplasts. Transportan has the highest uptake efficacy among the studied peptides, both in mammalian cells and plant protoplast. The internalisation of CPPs by plant protoplasts may open up a new effective method for transfection in plants.

Galanin is a 29/30 amino acid neuropeptide that has been shown to impair learning and memory task performance and also have roles in somatosensation, stress responses, sexual behavior, and feeding regulation. However, little is known about galanin involvement in higher cognitive processes, especially executive processes. Perseveration is a classic sign of frontal cortex damage and failure of executive control. Galanin has been shown to disrupt the performance of maze delayed alternation tasks and the operant, spatial delayed nonmatch-to-position (DNMTP) working memory task, tests especially sensitive to perseverative responding. To better understand this potential involvement of galanin in executive control, the present study tested the hypothesis that galanin induces perseveration. The first experiment examined the effects of galanin (10, 20 microg i.c.v.) on the performance of a simple operant response alternation task in which stimuli were assigned to one of two spatially distinct locations to produce extended sequences of presentations to one location, separated by a 10-s intertrial interval. The second experiment looked at the effects of galanin (5, 20 microg i.c.v.) on the performance of non-delayed match-to-position and nonmatch-to-position conditional discrimination operant tasks in which a minimal 1.0 s time interval separated responses. Finally, the effects of galanin (10, 20 microg i.c.v.) on delayed match-to-position (DMTP) performance were examined to determine whether response alternation (i.e., nonmatching) was critical to observing a galanin-induced impairment in this task. Galanin reduced the rate of trial completion in all the tasks, but did not alter simple or conditional discrimination accuracy. Galanin (10 microg) impaired DMTP performance in a delay-independent manner. Together, these data suggest that galanin does not produce perseveration, but are consistent with a galanin-induced decrease in reinforcer strength.

Thiol-containing compounds are central actors in many biochemical and pharmacological reactions. The response of cells to any stress (including cell division and apoptosis) involves changes in thiol content as they are consumed to protect cells via different actions (direct modification/regulation of biomolecules, antioxidativity, detoxification, signal transmission). The story of glutathione, the basic intracellular thiol-containing compound, ranges throughout different scientific fields. The importance of this biomolecule is highly impressive. Reduced glutathione (GSH) is a principal actor in many physiological and pharmacological reactions. There are about 60 000 entries under “glutathione” found in the Medline database. The aim of this short review is to characterize glutathione and show that due to an intriguing and multifaceted biofunctionality in the human body this tripeptide itself, as well as its analogues, belongs to the group of molecules looking for broad clinical use (besides being excellent antioxidants). This information might draw more attention to the discovery and investigation of glutathione system supporting/relating substances with a substantial clinical impact.

The chimeric peptide M617, galanin(1–13)-Gln14-bradykinin(2–9)amide, is a novel galanin receptor ligand with increased subtype specificity for GalR1 and agonistic activity in cultured cells as well as in vivo. Displacement studies on cell membranes expressing hGalR1 or hGalR2 show the presence of a high affinity binding site for M617 on GalR1 (Ki=0.23±.12 nM) while lower affinity was seen towards GalR2 (Ki=5.71±1.28 nM) resulting in 25-fold specificity for GalR1. Activation of GalR1 upon stimulation with M617 is further confirmed by internalization of a GalR1-EGFP conjugate. Intracellular signaling studies show the ability of M617 to inhibit forskolin stimulated cAMP formation with 57% and to produce a 5-fold increase in inositol phosphate (IP) accumulation. Agonistic effects on signal transduction are shown on both receptors studied after treatment with M617 in the presence of galanin. In noradrenergic locus coeruleus neurons, M617 induces an outward current even in the presence of TTX plus Ca2+, high Mg2+, suggesting a postsynaptic effect. Intracerebroventricular (i.c.v.) administration of M617 dose-dependently stimulates food uptake in rats while, in contrast, M35 completely fails to affect the feeding behavior. Spinal cord flexor reflex is facilitated by intrathecal (i.t.) administration of M617 as well as galanin with no significant change upon pre-treatment with M617. M617 dose dependently antagonizes the spinal cord hyperexcitablility induced by C-fiber conditioning stimulus and does neither enhance nor antagonize the effect of galanin. These data demonstrate a novel galanin receptor ligand with subtype specificity for GalR1 and agonistic activity, both in vitro and in vivo.

Cell-penetrating peptides (CPPs) are short peptides of less than 30 amino acids that are able to penetrate cell membranes and translocate different cargoes into cells. The only common feature of these peptides appears to be that they are amphipathic and net positively charged. The mechanism of cell translocation is not known but it is apparently receptor and energy independent although, in certain cases, translocation can be partially mediated by endocytosis. Cargoes that are successfully internalized by CPPs range from small molecules to proteins and supramolecular particles. Most CPPs are inert or have very limited side effects. Their penetration into cells is rapid and initially first-order, with half-times from 5 to 20 min. The size of smaller cargoes does not affect the rate of internalization, but with larger cargoes, the rate is substantially decreased. CPPs are novel vehicles for the translocation of cargo into cells, whose properties make them potential drug delivery agents, of interest for future use.

Different glutathione analogues have potential to maintain or increase tissue glutathione level and to scavenge the reactive oxygen species. We designed and synthesized a novel non-toxic glutathione analogue, named UPF1, which possessed 60-fold higher hydroxyl radical scavenger efficiency in vitro, compared with glutathione itself, and investigated the effects of UPF1 on a four-vessel occlusion model of rats. The UPF1 was administered via the jugular vein in two separate experiments at two time points: 20 min before global brain ischemia and immediately before reperfusion. In both cases the number of pyramidal cells surviving in the subfield of CA1 at the dorsal hippocampus in the UPF1-treated groups of rats was twice as high as in the vehicle group.

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.

The activation of nuclear factor kappaB (NFkappaB) is a key event in immune and inflammatory responses. In this study, a cell-penetrating transport peptide, transportan (TP) or its shorter analogue TP 10, was used to facilitate the cellular uptake of an NFkappaB decoy. Peptide nucleic acid (PNA) hexamer or nonamer was linked to the transport peptide by a disulfide bond. NFkappaB decoy oligonucleotide consisted of a double-stranded consensus sequence corresponding to the kappaB site localized in the IL-6 gene promoter, 5'-GGGACTTTCCC-3', with a single-stranded protruding 3'-terminal sequence complementary to the PNA sequence was hybridized to the transport peptide-PNA construct. The ability of the transport peptide-PNA-NFkappaB decoy complex to block the effect of interleukin (IL)-1beta-induced NFkappaB activation and IL-6 gene expression was analyzed by electrophoretic mobility shift assay and reverse transcriptase-polymerase chain reaction in rat Rinm5F insulinoma cells. Preincubation with transport peptide-PNA-NFkappaB decoy (1 microM, 1 h) blocked IL-1beta-induced NFkappaB-binding activity and significantly reduced the IL-6 mRNA expression. The same concentration of NFkappaB decoy in the absence of transport peptide-PNA had no effect even after longer incubations. Our results showed that binding of the oligonucleotide NFkappaB decoy to the nonamer PNA sequence resulted in a stable complex that was efficiently translocated across the plasma membrane.

The neuropeptide galanin has been implicated in inhibiting seizures and protecting hippocampal neurons from excitotoxic injury. In the hippocampus galanin acts through two receptor subtypes, GalR1, expressed in CA1, and GalR2, abundant in dentate gyrus. We developed an approach to induce and to study selective semichronic knockdown of GalR2 in the rat hippocampus. A 50% reduction of GalR2 binding was achieved by continuous infusion of complementary peptide nucleic acid antisense oligonucleotide into the dentate gyrus. This resulted in an increase in the severity of self-sustaining status epilepticus induced by electrical stimulation of the perforant path, induced mild neuronal injury in the dentate hilus, augmented seizure-induced hilar injury and inhibited seizure-induced neurogenesis in the subgranular zone of the dentate gyrus. Our data suggest that in the dentate gyrus, galanin, acting through GalR2, inhibits seizures, promotes viability of hilar interneurons and stimulates seizure-induced neurogenesis. These results are important for understanding the role of galanin and galanin receptor subtypes in the hippocampus both under normal conditions and in excitotoxic injury.

A novel carrier system that originates from membrane shuttling proteins such as the Drosophila homeobox protein Antennapedia, the HIV-1 transcriptional factor TAT and VP22 from HSV-1 has advantages for targeted delivery compared with standard translocation techniques. This transport system is mediated by so-called cell-penetrating peptides, which consist of short peptide sequences that rapidly translocate large molecules into the cell interior in a seemingly energy- and receptor-independent manner. Cell-penetrating peptides have low toxicity and a high yield of delivery and in the future might become a widely used tool in the field of gene regulation.

Cellular machinery is protected from the surrounding by two-layer lipid membrane that is impermeable for most substances unnecessary for cellular metabolism. Unfortunately, from a cellular point of view, most new generation drugs, designed to act on gene regulation and transcription, are also considered to be unnecessary for metabolism and therefore showing poor, if any, intracellular localization. To overcome this obstacle, several chemical and physical methods have been developed, improving the uptake, but, on the other hand, also showing some unwanted side effects or limitations for in vivo applications. This dictates the continuing need for improved drug delivery and one way seems to be the relatively new class of compounds – cell-penetrating peptides (CPPs). Discovered approximately a decade ago, the content of this class is growing rapidly, containing now more than 100 compounds, which shows the intensity of work in this field. CPPs have already been shown to translocate cellular membranes in an unknown, seemingly receptor-independent and non-endocytotic manner. Moreover, they are able to deliver cargoes exceeding their own size up to 100-fold into a cellular milieu both in vitro and in vivo. The variety of different cargoes includes, but is not limited to: DNA, antisense PNA, oligonucleotides and small proteins. Recent data argues though that endocytosis is involved and contributes in some cases to the main part of the translocation. This review summarizes data on mechanisms of cell-penetrating peptides.

Antimicrobial drug action is limited by both microbial and host cell membranes. Microbes stringently exclude the entry of most drugs, and mammalian membranes limit drug distribution and access to intracellular pathogens. Recently, cell-penetrating peptides (CPPs) have been developed as carriers to improve mammalian cell uptake. Given that CPPs are cationic and often amphipathic, similar to membrane active antimicrobial peptides, it may be possible to use CPP activity to improve drug delivery to microbes. Here, two CPPs, TP10 and pVEC, were found to enter a range of bacteria and fungi. The uptake route involves rapid surface accumulation within minutes followed by cell entry. TP10 inhibited Candida albicans and Staphylococcus aureus growth, and pVEC inhibited Mycobacterium smegmatis growth at low micromolar doses, below the levels that harmed human HeLa cells. Therefore, although TP10 and pVEC entered all cell types tested, they preferentially damage microbes, and this effect was sufficient to clear HeLa cell cultures from noninvasive S. aureus infection. Also, conversion of the cytotoxicity indicator dye SYTOX Green showed that TP10 causes rapid and lethal permeabilization of S. aureus and pVEC permeabilizes M. smegmatis, but not HeLa cells. Therefore, TP10 and pVEC can enter both mammalian and microbial cells and preferentially permeabilize and kill microbes.

We have examined the effect of systemically administered galnon, a novel low-molecular weight agonist of galanin receptors, on neuropathic pain-like behaviors in rats after photochemically induced partial nerve injury. Galnon is a galanin receptor ligand with moderate affinity to spinal cord membranes ( K D of 6±0.6 μM). While intraperitoneally applied galnon produced no significant effect on mechanical or cold hypersensitivity, it dose-dependently prolonged heat withdrawal latency in nerve-injured rats. The effect of galnon was more potent on the injured side which has significantly shorter latency than the contralateral side. The anti-hyperalgesic effect of galanon was prevented by intrathecal M35, a galanin receptor antagonist. No side effects, such as sedation or motor impairment, were seen following systemic galnon treatment at the doses used. It is concluded that systemic galnon alleviated heat-hyperalgesic response in rats with partial sciatic nerve injury. This effect was likely to be mediated by activation of spinal galanin receptors.

The neuropeptide galanin (Gal) and its receptors (GalR1, GalR2, and GalR3) are expressed in spinal cord. We have characterized the pharmacology of the antinociceptive effects of intrathecally (i.t.) administered galanin and its analogs in the formalin test in rats, using an automated flinch detection system. Intrathecal injection of rat galanin (Gal(1-29)) or human galanin (Gal(1-30)) produced a dose-dependent inhibition of formalin-evoked flinching in phase 2, but not in phase 1. Relative potency of galanin homologs is Gal(1-29) >or= Gal(1-30) > galanin-like peptide(1-24) >or= Gal(2-11) = Gal (3-29) (an inactive analog). Galanin(1-29) and Gal(1-30) are both high-affinity agonists to GalR1/R2, whereas Gal(2-11) is a GalR2 receptor agonist. Our data suggest that i.t. galanin-produced antinociception is mediated by activation of GalR1 receptors. When comparing antinociceptive effects of i.t. Gal(1-29) to morphine and to 2-amino-5-phosphonopentanoic acid (AP-5, an N-methyl-d-aspartate antagonist), Gal(1-29) is of intermediate potency between these two analgesic agents based on the ED(50) values. An isobolographic analysis showed synergy between Gal(1-29) and morphine and between Gal(1-29) and AP-5 on the second phase. Fixed ratio dose combinations of morphine and Gal(1-29), or AP-5 and Gal(1-29) produced significantly greater antinociception than predicted from simple additivity. In summary, the present findings reveal that 1) spinal galanin produces a reliable inhibition of formalin-induced facilitated nociceptive processing, an effect possibly mediated by GalR1 receptors; and 2) galanin potentiates i.t. morphine and AP-5-induced antinociception.

Previous studies revealed the importance of the third intracellular loop of glucagon-like peptide-1 receptor (GLP-1R) in coupling to G(s) and G(i1) proteins. In order to further study the signaling mechanisms of GLP-1R, we tested three peptides, corresponding to the sequences of the first (IC(1)), the second (IC(2)), and the third (IC(3)) intracellular loop of GLP-1R, for their interactions with heterotrimeric G-proteins of different types (G(alphas), G(alphao), G(alphai1), and G(alpha11) plus G(beta1gamma2)) overexpressed in sf9 cells. IC(3) peptide powerfully stimulates all types of tested G-proteins, whereas IC(1) and IC(2) peptides show differential effects on G-proteins. Both IC(1) and IC(2) peptides activate G(s) and cooperate with IC(3) peptide in its stimulation. G(o) is not affected by IC(1) and IC(2). G(i1) and G(11) are not affected by IC(1), but are activated by IC(2), which in activation cooperates with IC(3). We suggest that GLP-1R is not coupled only to G(s) and G(i1), as shown previously, but also to G(o) and G(11). IC(3) loop is the main switch that mediates signaling via GLP-1R to G-proteins, while IC(1) and IC(2) loops are important in discrimination between different types of G-proteins.

Cell membranes act as protective walls to exclude most molecules that are not actively imported by living cells. This is an efficient way for a cell to prevent uncontrolled influx or efflux of solutes, which otherwise would be harmful to it. Only compounds within a narrow range of molecular size, polarity and net charge are able to diffuse effectively through cell membranes. In order to overcome this barrier for effective delivery of membrane-impermeable molecules, several chemical and physical methods have been developed. These methods, e.g. electroporation, and more recent methods as cationic lipids/liposomes, have been shown to be effective for delivering hydrophobic macromolecules. The drawbacks of these harsh methods are, primarily, the unwanted cellular effects exerted by them, and, secondly, their limitation to in vitro applications. The last decade's discovery of cell-penetrating peptides translocating themselves across cell membranes of various cell lines, along with a cargo 100-fold their own size, via a seemingly energy-independent process, opens up the possibility for efficient delivery of DNA, antisense peptide nucleic acids, oligonucleotides, proteins and small molecules into cells both in vitro and in vivo.

1. The role of individual residues in the 8-18 helix of CGRP(8-37) in promoting high-affinity binding to CGRP(1) receptors expressed on rat L6 and human SK-N-MC cells has been examined. The relative potencies of various derivatives were estimated from their ability to inhibit the human alphaCGRP-mediated increase in cyclic AMP production and the binding of [(125)I]-human alphaCGRP. 2. Arg(11) and Arg(18) were replaced by serines to give [Ser(11,18)]CGRP(8-37). These bound with pKi values

The receptor for calcitonin-gene-related peptide (CGRP) is a heterodimer formed by calcitonin-receptor-like receptor (CRLR), a type II (family B) G-protein-coupled receptor, and receptor-activity-modifying protein 1 (RAMP1), a single-membrane-pass protein. It is likely that the first seven or so amino acids of CGRP (which form a disulphide-bonded loop) interact with the transmembrane domain of CRLR to cause receptor activation. The rest of the CGRP molecule falls into three domains. Residues 28–37 and 8–18 are normally required for high-affinity binding, while residues 19–27 form a hinge region. The 28–37 region is almost certainly in direct contact with the receptor; 8–18 may make additional receptor contacts or may stabilize an appropriate conformation of 28–37. It is likely that these regions of CGRP interact both with CRLR and with the extracellular domain of RAMP1.

Galanin is a neuropeptide with a wide variety of biological functions, including that of a strong endogenous anticonvulsant. No nonpeptide ligands, capable of activating galanin receptors, are available today. Based on known pharmacophores of galanin, a combinatorial library was designed, synthesized, and screened at the rat hippocampal galanin receptor. A low molecular weight galanin receptor agonist, 7-((9-fluorenylmethoxycarbonyl)cyclohexylalanyllysyl)amino-4-methylcoumarin (galnon) was found to displace 125 I-galanin with micromolar affinity at Bowes cellular and rat hippocampal membranes. Autoradiographic binding assay on rat spinal cord sections confirmed the ability of galnon to displace 125 I-galanin from its binding sites. Galnon inhibited adenylate cyclase activity, suggesting an agonist action at galanin receptors. When injected i.p. galnon reduced the severity and increased the latency of pentylenetetrazole-induced seizures in mice and reversed the proconvulsant effects of the galanin receptor antagonist M35, injected into a lateral ventricle. Intrahippocampal injection of galnon also shortened the duration of self-sustaining status epilepticus in rats, confirming its agonist properties in vivo . Pretreatment of rats with antisense peptide nucleic acid targeted to galanin receptor type 1 mRNA abolished the effect of galnon, suggesting mediation of its anticonvulsant properties through this receptor subtype. These findings introduce a systemically active nonpeptide galanin agonist anticonvulsant.

The expression of galanin receptor-1 (GALR1) was investigated in the rat trigeminal ganglion by using immunocytochemistry and in situ hybridization. In addition, the regional distribution of GALR1-immunoreactive pulpal nerves and their ultrastructure were examined in the molar teeth. In the trigeminal ganglion, the immunoreactivity for GALR1 was recognizable in about 30% of the total number of neurons. Most of the cell bodies were small to medium in size. Analysis of serially cut sections alternately stained with GALR1 and galanin antisera demonstrated that some GALR1-positive cells displayed immunoreactivity for galanin. In situ hybridization analysis, expression of GALR1 mRNA was detected in trigeminal ganglion cells. The cell size distribution was similar to that of GALR1-immunoreactive cells. In the dental pulp, a small number of nerve fibers displayed immunoreactivity for GALR1. The labeled fibers formed terminal arbors in the coronal pulp around and within the odontoblast cell layer, but never penetrated into the predentin and dentin. Ultrastructurally, GALR1 immunoreactivity in the dental pulp was confined to the axoplasm of unmyelinated nerve fibers. The present study provided new evidence that unmyelinated primary afferents innervating dental pulp possessed galanin receptor, and suggests the existence of nociceptive primary afferents functioning as autocrine cells.

Cationic amphiphilic peptides stimulate secretion via a receptor-independent action upon G proteins [1]. We have previously utilized chimeric analogs of mastoparan (MP), including galparan (galanin(1–13)-MP [2]), as molecular probes of secretion [3, 4]. Here, we further resolve the structure-activity relationship of peptidyl secretagogs, including rationally designed chimeric MP analogs. The secretory efficacies of 10 MP analogs were significantly higher than 45 unrelated basic peptides. Comparative studies identified MP analogs that are differential secretagogs for 5-hydroxytryptamine (5-HT) and β-hexosaminidase. Peptide-induced activation of phospholipase D (PLD), an enzyme intimately involved in regulated exocytosis [5], correlated with the secretion of β-hexosaminidase but not 5-HT. Thus, these data indicate that different mechanisms are responsible for the exocytosis of 5-HT and β-hexosaminidase, respectively. Moreover, mastoparan analogs are novel tools for probing the molecular details of exocytosis and other biological phenomena.

Cell-penetrating peptides (CPPs) have been used as vehicles to deliver various cargos into cells and are promising as tools to deliver therapeutic biomolecules such as oligonucleotides both in vitro and in vivo. CPPs are positively charged and it is believed that CPPs deliver their cargo in a receptor-independent manner by interacting with the negatively charged plasma membrane and thereby inducing endocytosis. In this study we examine the mechanism of uptake of several different, well known, CPPs that form complexes with oligonucleotides. We show that these CPP:oligonucleotide complexes are negatively charged in transfection-media and their uptake is mediated by class A scavenger receptors (SCARA). These receptors are known to promiscuously bind to, and mediate uptake of poly-anionic macromolecules. Uptake of CPP:oligonucleotide complexes was abolished using pharmacological SCARA inhibitors as well as siRNA-mediated knockdown of SCARA. Additionally, uptake of CPP:oligonucleotide was significantly increased by transiently overexpressing SCARA. Furthermore, SCARA inhibitors also blocked internalization of cationic polymer:oligonucleotide complexes. Our results demonstrate that the previous held belief that CPPs act receptor independently does not hold true for CPP:oligonucleotide complexes, as scavenger receptor class A (SCARA) mediates the uptake of all the examined CPP:oligonucleotide complexes in this study.

Peptides and peptide-cargo complexes have been used for drug delivery and gene therapy. One of the most used delivery vectors are cell-penetrating peptides, due to their ability to be taken up by a variety of cell types and deliver a large variety of cargoes through the cell membrane with low cytotoxicity. In vitro and in vivo studies have shown their possibility and full effectiveness to deliver oligonucleotides, plasmid DNA, small interfering RNAs, antibodies, and drugs. We report in this review some of the latest strategies for peptide-mediated delivery of nucleic acids. It focuses on peptide-based vectors for therapeutic molecules and on nucleic acid delivery. In addition, we discuss recent applications and clinical trials.

The binding affinity of a series of cell-penetrating peptides (CPP) was modeled through docking and making use of the number of intermolecular hydrogen bonds, lipophilic contacts, and the number of sp3 molecular orbital hybridization carbons. The new ranking of the peptides is consistent with the experimentally determined efficiency in the downregulation of luciferase activity, which includes the peptides’ ability to bind and deliver the siRNA into the cell. The predicted structures of the complexes of peptides to siRNA were stable throughout 10 ns long, explicit water molecular dynamics simulations. The stability and binding affinity of peptide-siRNA complexes was related to the sidechains and modifications of the CPPs, with the stearyl and quinoline groups improving affinity and stability. The reranking of the peptides docked to siRNA, together with explicit water molecular dynamics simulations, appears to be well suited to describe and predict the interaction of CPPs with siRNA.

Despite the extensive research in the field of CPPs' cell entry the exact mechanisms underlying their cellular uptake and the role of involved cell surface molecules in the internalization process have remained controversial. The present study focused on the interactions between CPPs and plasma membrane compounds using giant plasma membrane vesicles (GPMVs). GPMVs have shown to be a suitable model to study the translocation of CPPs across the plasma membrane in conditions lacking endocytosis. Our results show that higher cholesterol content and tighter packing of membrane predominantly reduce the accumulation of transportan, TP10 and model amphipathic peptide (MAP) in vesicles, indicating that the internalization of CPPs takes place preferentially via the more dynamic membrane regions. The partial digestion of membrane proteins from GPMVs' surface, on the other hand, drastically reduced the accumulation of nona-arginine and Tat peptide into vesicles, suggesting that proteins play a crucial role in the uptake of arginine-rich CPPs.

Galanin is a neuroendocrine peptide involved in the regulation of feeding, pain, sexual behavior, learning, and memory. The recent discovery, that galanin antagonized excitatory glutamatergic neurotransmission in the hippocampus, provided a rationale for its possible antiepileptic effects. Here we summarize the data on the effects of galanin on seizure activity in several animal models of epilepsy. Pharmacological and molecular biological evidence suggest potent anticonvulsant effects of galanin. Exogenous administration of galanin receptor agonists attenuated seizures, whereas application of galanin receptor antagonists potentiated seizure expression. Genetically engineered mice, with either deletion or overexpression of galanin gene, showed altered resistance to seizures, which was in direct correlation with galanin gene expression. Possible mechanisms of the anticonvulsant action of galanin include its effects on synaptic potentiation in hippocampal circuits and inhibition of the release of the excitatory neurotransmitter glutamate from principal hippocampal neurons.

Cellular translocation into a human Bowes melanoma cell line was investigated and compared for penetratin and pIsl, two peptides that correspond to the third helices of the related homeodomains, from the Antennapedia transcription factor of Drosophila and the rat insulin-1 gene enhancer protein, respectively. Both biotinylated peptides internalized into the cells with similar efficacy, yielding an analogous intracellular distribution. When a large cargo protein, 63 kDa avidin, was coupled to either peptide, efficient cellular uptake for both the peptide-protein complexes was observed. The interactions between each peptide and SDS micelles were studied by fluorescence spectroscopy and acrylamide quenching of the intrinsic tryptophan (Trp) fluorescence. Both peptides interacted strongly and almost identically with the membrane mimicking environment. Compared to penetratin, the new transport peptide pIsl has only one Trp residue, which simplifies the interpretation of the fluorescence spectra and in addition has a native Cys residue, which may be used for alternative coupling reactions of cargoes of different character.

In this work, we studied a novel chimeric peptide, M242, galanin(1-13)-[D-Trp(32)]-neuropeptide Y(25-36)amide, and examined its properties in comparison with its parent peptide, M32, galanin(1-13)-neuropeptide Y(25-36)amide, a previously known high-affinity ligand for galanin receptors, and galanin itself. Binding assays performed in Bowes cells known to express human galanin receptor type 1 (hGalR1) and in Chinese hamster ovary cells overexpressing human galanin receptor type 2 (hGalR2) revealed that all three ligands had comparable affinities: at hGalR11 nM and at hGalR210 nM. However, in rat hippocampal membranes M242 had a 24-fold lower affinity than galanin (9.4 vs. 0.4 nM) and 134-fold lower affinity than M32 (9.4 vs. 0.07 nM). In the same tissue, we also examined the effects of these peptides on adenylate cyclase activity. M32 showed a weak antagonistic behaviour but M242 acted as a potent biphasic regulator of adenylate cyclase. In conclusion, we present and characterise a new peptide M242, which could be a useful tool in studies of galaninergic signalling.

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.

Ligand binding to neuropeptide Y (NPY) receptors Y1, Y2, Y4, and Y5 from guinea-pig was investigated using the two NPY-galanin hybrids M32 (galanin1-13-NPY25–36-amide) and M242 ([D-Trp 32 ]M32). The affinity of M32 for Y1, Y2, and Y4 receptors was 13, 4, and 30 nM, respectively, similar to that of NPY18–36 and NPY22–36 but 40-fold to 300-fold lower than the affinity of intact porcine NPY. M242 bound to the Y1, Y2, and Y4 receptors with 9-fold to 20-fold lower affinity than did M32. The affinities of M32 and M242 for Y5 were 400 and 800 nM, respectively. Thus, M32 seems to gain affinity relative to both of its constituent peptide portions although the NPY25–36 part may be sufficient for NPY-receptor recognition, especially at the Y2 receptor. This suggests that the galanin portion of M32 influences and/or stabilizes the conformation of the NPY portion, similar to the effect seen for the NPY portion of M32 in binding to galanin receptors.

The p53 transcription factor is either latent or activated through multi-site phosphorylation and acetylation of the negative regulatory region in its C-terminal domain (CTD). How CTD modifications activate p53 binding to target DNA sequences via its core domain is still unknown. It has been proposed that nonmodified CTD interacts either with the core domain or with DNA preventing binding of the core domain to DNA and that the fragments of the CTD regulatory region activate p53 by interfering with these interactions. We here characterized the sequence and target specificity of p53 activation by CTD fragments, interaction of activating peptides with p53 and target DNA, and interactions of "latent" p53 with DNA by a band shift assay and by fluorescence correlation spectroscopy. In addition to CTD fragments, several long basic peptides activated p53 and also transcription factor YY1. These peptides and CTD aggregated target DNA but apparently did not interact with p53. The potency to aggregate DNA correlated with the ability to activate p53, suggesting that p53 binds to target sequences upon interactions with tightly packed DNA in aggregates. Latent full-length p53 dissociated DNA aggregates via its core and CTD, and this effect was potentiated by GTP. Latent p53 also formed complexes via both its core and CTD with long nontarget DNA molecules. Such p53-DNA interactions may occur if latent p53 binding to DNA via CTD prevents the interaction of the core domain with target DNA sites but not with nonspecific DNA sequences.

It has previously been shown that the GLP-1 receptor is primarily coupled to the adenylate cyclase pathway via activation of Galpha(s) proteins. Recent studies have shown that the third intracellular loop of the receptor is important in the stimulation of cAMP production. We have studied the effect of three synthetic peptide sequences derived from the third intracellular loop of the GLP-1 receptor on signal transduction in Rin m5F cell membranes. The whole third intracellular loop strongly stimulates both pertussis toxin and cholera toxin-sensitive G proteins, while the N-terminal half exclusively stimulates cholera toxin-sensitive G proteins and the C-terminal half only stimulates pertussis toxin-sensitive G-proteins as demonstrated by measurements of GTPase activity. These data confirm that the principal stimulatory G-protein interaction site resides in the third intracellular loop, but also suggest that the GLP-1 receptor is not only coupled to the Galpha(s) but also to the Galpha(i)/Galpha(o) type of G proteins and that distinct domains within the third intracellular loop are responsible for the activation of the different G-protein subfamilies.

One of the pathological hallmarks of Alzheimer's disease (AD) is the presence of amyloid plaques. The main constituent of the amyloid plaques is the amyloid beta-peptide (A beta) shown to activate glial cells in vitro. A growing body of evidence suggests that these cells contribute to neurotoxicity through production of inflammatory cytokines, chemokines, and neurotoxic substances, such as reactive oxygen species (ROS). In this study, mRNA levels of the inflammatory cytokines interleukin (IL)-1alpha and beta, and IL-6 were analysed by reverse transcriptase-polymerase chain reaction (RT-PCR) in rat primary mixed glial cells after treatment with A beta(25-35), a biologically active fragment of A beta peptide with neurotoxic properties. Clear morphological changes of the astrocytes, as well as proliferation and clustering of microglial cells were observed by light and immunofluorescence microscopy after 24 h treatment. Significant increases in IL-1alpha and IL-6 mRNA levels were detected after 24 and 72 h, whereas significantly increased levels of IL-1beta mRNA could only be detected after 4 h treatment. The most pronounced effect was seen on IL-6 mRNA expression, which increased approx two- to threefold after treatment. In addition, increased secretion of IL-6 was detected after 96 h exposure. Recently, association of IL-1alpha and IL-6 gene polymorphism with AD was reported, suggesting that these cytokines may play an important role in the development of the disease. The increased mRNA levels of IL-1alpha and IL-6 in parallel with the morphological changes in the mixed glial-cell cultures support that these cytokines may be involved in A beta-induced gliosis and in the pathogenesis of AD.