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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.

This study shows that lipopolysaccharide (LPS) induces a robust, concentration-and time-dependent increase in nitric oxide (NO) production in the murine neuroblastoma cell line N2a by increased expression of iNOS. However, in scrapie-infected N2a cells (ScN2a), the LPS-induced NO production was completely abolished. The absence of LPS-induced NO production in ScN2a was not due to inhibited enzymatic activity of iNOS but a complete inhibition of the LPS-induced iNOS gene expression as measured by Western blot and reverse transcription-polymerase chain reaction (RT-PCR). Furthermore, major changes in the protein tyrosine phosphorylation profile of scrapie-infected hypothalamic neurons (ScGT1-1) and ScN2a, compared to their uninfected counterparts were shown, by Western blot of whole cell extracts and of anti-phosphotyrosine immunoprecipitates, with major bands corresponding to120, 150 and 180 kD. Anti-phosphotyrosine blots of lectin-purified glycoproteins, indicated that some, but not all, of the proteins in the 120 kD band are glycosylated. Two phosphoproteins of ≈ 120 kDa were identified, the receptor tyrosine kinase, fibroblast growth factor 2 (FGFR2) and the cytoplasmic non-receptor tyrosine kinase proline-rich tyrosine kinase 2 (PYK2). In addition, scrapie-infection induces important alterations in the expression, binding and signalling of two neurotrophic receptors, the insulin-like growth factor-1 receptor (IGF-1R) and the insulin receptor (IR) in ScN2a, as compared to uninfected N2a cells. In ScN2a, the IGF-1R and IR protein levels were four- and two-fold increased, respectively, with an unexpected decrease in specific binding sites, as revealed by equilibrium binding studies. In the case of the IGF-1R, the apparent drop in binding sites was due to a seven-fold drop in IGF-1 affinity. Moreover, the IGF-1 stimulated IGF-1R tyrosine phosphorylation was increased in ScN2a when compared to the reduced affinity, possibly due to altered tyrosine kinase signalling in ScN2a. In the case of the IR, the binding affinity was unchanged, although insulin-stimulated IR tyrosine phosphorylation was increased.

A growing body of evidence suggests that an altered level or function of the neurotrophic insulin-like growth factor-1 receptor (IGF-1R), which supports neuronal survival, may underlie neurodegeneration. This study has focused on the expression and function of the IGF-1R in scrapie-infected neuroblastoma cell lines. Our results show that scrapie infection induces a 4-fold increase in the level of IGF-1R in two independently scrapie-infected neuroblastomas, ScN2a and ScN1E-115 cells, and that the increased IGF-1R level was accompanied by increased IGF-1R mRNA levels. In contrast to the elevated IGF-1R expression in ScN2a, receptor binding studies revealed an 80% decrease in specific125I-IGF-1-binding sites compared with N2a cells. This decrease in IGF-1R-binding sites was shown to be caused by a 7-fold decrease in IGF-1R affinity. Furthermore, ScN2a showed no significant difference in IGF-1 induced proliferative response, despite the noticeable elevated IGF-1R expression, putatively explained by the reduced IGF-1R binding affinity. Additionally, IGF-1 stimulated IGF-1Rβ tyrosine phosphorylation showed no major change in the dose-response between the cell types, possibly due to altered tyrosine kinase signaling in scrapie-infected neuroblastoma cells. Altogether these data indicate that scrapie infection affects the expression, binding affinity, and signal transduction mediated by the IGF-1R in neuroblastoma cells. Altered IGF-1R expression and function may weaken the trophic support in scrapie-infected neurons and thereby contribute to neurodegeneration in prion diseases.

Bacterial lipopolypolysaccharide (LPS)-induced fever involves induction of the proinflammatory cytokines interleukin (IL)-1 alpha, IL-1 beta, tumor necrosis factor-alpha (TNF-alpha), and IL-6, both in the periphery and in the brain. These molecules can induce expression of each other and also regulate expression of their own receptors in a complex manner. The functional hierarchy of these highly inducible proteins is therefore difficult to determine. Using mice strains carrying the null mutations of IL-1 beta, IL-1RI, IL-1RAcP, or IL-6, respectively, we show that LPS-induced fever involves IL-1 beta, which acts at a complex consisting of the type I IL-1 receptor and the IL-1RAcP. This action occurs prior to central IL-6 release, which has been shown to be a necessary component of fever responses induced by LPS, IL-1 beta, and also TNF-alpha. In the absence of IL-1 beta, as in IL-1 beta-deficient mice, LPS, IL-1 alpha, and IL-1 beta cause hyperresponsive fevers when exogenously applied. Murine TNF-alpha is a poor pyrogen in mice even when mice are kept at thermoneutral temperature (30 degrees C). TNF-alpha-mediated fever depends on central IL-6 expression.

Cell-penetrating peptides (CPPs) have proven utility for the highly efficient intracellular delivery of bioactive cargoes that include peptides, proteins, and oligonucleotides. The many strategies developed to utilize CPPs solely as pharmacokinetic modifiers necessarily requires them to be relatively inert. Moreover, it is feasible to combine one or multiple CPPs with bioactive cargoes either by direct chemical conjugation or, more rarely, as non-covalent complexes. In terms of the message-address hypothesis, this combination of cargo (message) linked to a CPP (address) as a tandem construct conforms to the sychnological organization. More recently, we have introduced the term bioportide to describe monomeric CPPs that are intrinsically bioactive. Herein, we describe the design and biochemical properties of two rhegnylogically organized monometic CPPs that collectively modulate a variety of biological and pathophysiological phenomena. Thus, camptide, a cell-penetrant sequence located within the first intracellular loop of a human calcitonin receptor, regulates cAMP-dependent processes to modulate insulin secretion and viral infectivity. Nosangiotide, a bioportide derived from endothelial nitric oxide synthase, potently inhibits many aspects of the endothelial cell morphology and movement and displays potent anti-angiogenic activity in vivo. We conclude that, due to their capacity to translocate and target intracellular signaling events, bioportides represent an innovative generic class of bioactive agents.

Resistance to chemotherapy limits the effectiveness of anti-cancer drug treatment. Here, we present a new approach to overcome the setback of drug resistance by designing a conjugate of a cell-penetrating peptide and the cytostatic agent methotrexate (MTX). Two different peptides, YTA2 and YTA4, were designed and their intracellular delivery efficiency was characterized by fluorescence microscopy and quantified by fluorometry. MTX was conjugated to the transport peptides and the ability of the peptide-MTX conjugates to inhibit dihydrofolate reductase, the target enzyme of MTX, was found to be 15 and 20 times less potent than MTX. In addition, in vitro studies were performed in a drug resistant cell model using the 100-fold MTX resistant breast cancer cells MDA-MB-231. At a concentration of 1 microM, the peptide-MTX conjugates were shown to overcome MTX resistance and kill the cells more efficiently than MTX alone. Estimated EC50's were determined for MTX, MTX-YTA2 and YTA2 to be 18.5, 3.8 and 20 microM, respectively. In summary, cell-penetrating peptide conjugation of MTX is a new way of increasing delivery, and thereby, the potency of already well-characterized therapeutic molecules into drug resistant tumour cells.

We have previously shown that ScN2a cells (scrapie-infected neuroblastoma N2a cells) express 2-fold- and 4-fold-increased levels of IR (insulin receptor) and IGF-1R (insulin-like growth factor-1 receptor) respectively. In addition, the IR α- and β-subunits are aberrantly processed, with apparent molecular masses of 128 and 85 kDa respectively, as compared with 136 and 95 kDa in uninfected N2a cells. Despite the 2-fold increase in IR protein, the number of 125I-insulin-binding sites was slightly decreased in ScN2a cells [Östlund, Lindegren, Pettersson and Bedecs (2001) Brain Res. 97, 161–170]. In order to determine the cellular localization of IR in ScN2a cells, surface biotinylation was performed, showing a correct IR trafficking and localization to the cell surface. The present study shows for the first time that neuroblastoma N2a cells express significant levels of IR–IGF-1R hybrid receptors, and in ScN2a cells the number of hybrid receptors was 2-fold higher than that found in N2a cells, potentially explaining the apparent loss of insulin-binding sites due to a lower affinity for insulin compared with the homotypic IR. Furthermore, the decreased molecular mass of IR subunits in ScN2a cells is not caused by altered phosphorylation or proteolytic processing, but rather by altered glycosylation. Enzymic deglycosylation of immunoprecipitated IR from N2a and ScN2a cells with endoglycosidase H, peptide N-glycosidase F and neuraminidase all resulted in subunits with increased electrophoretic mobility; however, the 8–10 kDa shift remained. Combined enzymic or chemical deglycosylation using anhydrous trifluoromethane sulphonic acid treatment ultimately showed that the IR α- and β-subunits from ScN2a cells are aberrantly glycosylated. The increased formation of IR–IGF-1R hybrids in ScN2a cells may be part of a neuroprotective response to prion infection. The degree and functional significance of aberrantly glycosylated proteins in ScN2a cells remain to be determined.

The interleukin-1 (IL-1) receptor antagonist (IL-1ra) is an endogenous antagonist that blocks the effects of the proinflammatory cytokines IL-1alpha and IL-1beta by occupying the type I IL-1 receptor. Here we describe transgenic mice with astrocyte-directed overexpression of the human secreted IL-1ra (hsIL-1ra) under the control of the murine glial fibrillary acidic protein (GFAP) promoter. Two GFAP-hsIL-1ra strains have been generated and characterized further: GILRA2 and GILRA4. These strains show a brain-specific expression of the hsIL-1ra at the mRNA and protein levels. The hsIL-1ra protein was approximated to approximately 50 ng/brain in cytosolic fractions of whole brain homogenates, with no differences between male and female mice or between the two strains. Furthermore, the protein is secreted, inasmuch as the concentration of hsIL-1ra in the cerebrospinal fluid was 13 (GILRA2) to 28 (GILRA4) times higher in the transgenic mice than in the control animals. To characterize the transgenic phenotype, GILRA mice and nontransgenic controls were injected with recombinant human IL-1beta (central injection) or lipopolysaccharide (LPS, peripheral injection). The febrile response elicited by IL-1beta (50 ng/mouse icv) was abolished in hsIL-1ra-overexpressing animals, suggesting that the central IL-1 receptors were occupied by antagonist. The peripheral LPS injection (25 micrograms/kg ip) triggered a fever in overexpressing and control animals. Moreover, no differences were found in LPS-induced (100 and 1,000 micrograms/kg ip; 1 and 6 h after injection) IL-1beta and IL-6 serum levels between GILRA and wild-type mice. On the basis of these results, we suggest that binding of central IL-1 to central IL-1 receptors is not important in LPS-induced fever or LPS-induced IL-1beta and IL-6 plasma levels.

Tumor necrosis factor-alpha (TNF-alpha) is a pleiotropic cytokine that orchestrates an array of local and systemic effects. For instance, acute exposure to a high dose of TNF-alpha results in septic shock and fever. We have used interleukin-1beta (IL-1beta)- and interleukin-6 (IL-6)-deficient mice, along with their wild-type equivalents, to define a role for TNF-alpha in fever. Briefly, the mice produced prostaglandin E2-dependent fevers in response to recombinant murine TNF-alpha (rmTNF-alpha). Furthermore, rmTNF-alpha (12 microgram/mouse ip) triggered a febrile response in IL-1beta-deficient mice as well as in their corresponding wild-type controls. In contrast, the IL-6-deficient mice were resistant to rmTNF-alpha (4.5 microgram/mouse ip), although their wild-type counterparts readily mounted a fever. In the IL-6-deficient mice, moreover, the febrile response to rmTNF-alpha could be restored by a central administration of rat recombinant IL-6 (500 ng/mouse icv). We thus conclude that TNF-alpha can trigger fever independent of IL-1beta but dependent on IL-6. We also suggest that central, rather than peripheral, IL-6 (plasma IL-6 was measured 2 h after pyrogenic challenge) is essential in TNF-alpha-induced fever.

The neuropathological outcome of metabolic, vascular or mechanical insults to the CNS depends on brain temperature; mild hypothermia is neuroprotective, whereas elevated brain temperature can cause additional neural damage. Studies in both animals and humans have shown that the core and the brain temperature do not always concur with one another. It is therefore important to develop methods for monitoring brain temperature. This paper describes an animal model (the rat) in which we have developed a method to measure, at thermoneutral ambient temperature, the brain and core temperature concomitantly, during different drug treatments. We have used this animal model to study body temperature during fever (induced by human recombinant IL-1β, 5 µg/kg, i.p.), stress-induced hyperthermia (handling of the animal), hypothermia (induced by (±)-8-hydroxy-2-dipropylaminotetralin hydrobromide, 0.5 mg/kg, i.p.) and sleep (non-induced, other than by light and diurnal variation). We show that the thermal curves are similar in the brain and the peritoneum, independent of the thermal state.