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The interaction of heme or heme-artemisinin adducts (heme-art) with different peptides mimicking repeat sequences of the Histidine-Rich-Protein-II of Plasmodium falciparum (PfHRP-II) was investigated. The pseudo-first order rate constants of the coordination of heme or heme-art onto a histidine rich peptide, used as a mimic of PfHRP-II putative heme binding sequence, are of the same order of magnitude, namely 42 and 14 s(-1), respectively. Despite the intrinsic reactivity of the carbonyl at C10 of heme-art toward a hydroxyl function, a peptide containing a serine or threonine residue does not readily react with heme-art adducts. Therefore, a much higher affinity of heme-art compared to heme toward PfHRP-II, if so, must be induced by a specific interaction or a chemical reaction, these phenomena being both due to the tertiary structure of the parasite protein itself.

Several neuropeptide FF (NPFF)-related peptides, known as modulators of the opioid system, have been previously characterized in bovine and rodent brain. Reverse-phase high pressure liquid chromatography (HPLC) fractions of a human with normal pressure hydrocephalus cerebrospinal fluid (CSF), co-migrating with NPFF-related synthetic peptides, were characterized by capillary HPLC coupled on-line to nanospray ion trap tandem mass spectrometry. Two peptides present in the pro-NPFF(A) precursor, NPAF (AGEGLNSQFWSLAAPQRF-NH2) and NPSF (SLAAPQRF-NH2), were identified. The monitoring of NPFF-related peptides in human CSF can be helpful to understand their roles in pain sensitivity.

Peptides which should be generated from the neuropeptide FF (NPFF) precursor were identified in mouse and rat spinal cord, by using reverse phase high pressure liquid chromatography with radioimmunoassay and electrospray mass spectrometry detection. In both species, two octapeptides, NPFF (Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-amide) and NPSF (Ser-Leu-Ala-Ala-Pro-Gln-Arg-Phe-amide) were identified but a longer peptide NPA-NPFF (Asn-Pro-Ala-Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-amide) was present at the highest concentration in rat spinal cord. In mouse, the homologous peptide, SPA-NPFF (Ser-Pro-Ala-Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-amide) was not detected. Both peptides NPFF and NPSF reverse morphine-induced analgesia in the tail flick test. Our data reveal species differences in the maturation of NPFF precursor.

We have compared the affinities and anti-opioid activities of the different peptides putatively produced by the rat NPFF precursor, NPAFLFQPQRF-NH(2) (NPA-NPFF) and EFWSLAAPQRF-NH(2) (EFW-NPSF), with those already identified in nervous tissue, FLFQPQRF-NH(2) (NPFF) and SLAAPQRF-NH(2) (NPSF). NPFF and NPA-NPFF exhibit a high affinity (0.34 and 0.14 nM, respectively) for [(125)I]1DMe binding sites of the rat spinal cord. In contrast, EFW-NPSF displays an affinity 13 times higher than NPSF (1.99 and 9.5 nM, respectively). In rat dorsal raphe neurones, EFW-NPSF, NPFF, and NPA-NPFF maximally reduce the inhibitory effect of nociceptin on the [Ca(2+)](i) transients triggered by depolarization by 39, 31, and 58%, respectively. NPSF is inactive in the same test. We conclude that NPA-NPFF and EFW-NPSF are likely to be the physiologically active neurotransmitters in rat brain., (Copyright 2000 Academic Press.)

Peptide vaccines based on the use of MHC class I restricted epitopes are currently assayed for anti-tumor and anti-viral immunotherapy. With the aim of designing minimally modified, peptidase-resistant analogs, we developed a rational approach based on a detailed understanding of the degradation mechanism of peptides in serum. Degradation of murine tumor antigen P198 and human tumor antigen MAGE-3.A1 was followed by on line high performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS). This method provided high precision and sensitivity for rapid and direct analysis of degradation fragments in a complex mixture and, very importantly, precise identification of transient degradation fragments present at low concentrations. The design of structurally modified analogs, and the analysis of their degradation by on-line HPLC/ESI-MS, allowed us to to demonstrate the efficiency of local modifications in the protection of a given peptide bond towards a specific peptidase activity.

Polyclonal antibodies have been raised against the peptide 28-38 of the beta-subunit of the tubulin heterodimer in order to study the accessibility of this region in the tubulin heterodimer and in various tubulin assemblies. These antibodies were specific for all beta-tubulin subunits, except for beta'-tubulin isotypes, and did not recognize the alpha-tubulin subunit. The 28-38 region does not play a role in the interaction between the alpha- and beta-subunits since it was accessible to the antibodies on the native heterodimer. The accessibility of the antibodies was not modified by several microtubular poisons. In contrast, in all tubulin assemblies obtained in the presence of microtubular associated proteins, the region 28-38 was not available to the antibodies. These antibodies did not react with microtubules or tubulin spirals assembled either from microtubule proteins or from pure tubulin when these tubulin assemblies were probed in the absence of free tubulin after centrifugation on glass coverslips. In addition, antibodies failed to interact with the microtubule cytoskeleton in cultured Ptk2 cells indicating that the 28-38 region of beta-tubulin is also protected in cellular structures. These observations suggest that the 28-38 region of the beta-tubulin subunit is either located in a zone of interaction between two successive tubulin dimers within a protofilament or hidden by an allosteric conformational change which occurs during tubulin assembly.

Abstract: By using an optimized [35S]GTPγS binding assay, the functional activities (potency and efficacy) of peptides belonging to three members of the RFamide family; Neuropeptide FF (NPFF), prolactin-releasing peptide (PrRP) and 26RFamide, were investigated on NPFF1 and NPFF2 receptors stably expressed in Chinese Hamster Ovary (CHO) cells. Despite their large differences in affinity and selectivity, all analogues tested behaved as agonists toward NPFF1 and NPFF2 receptors. High NaCl concentration in the assay strongly increased the efficacy toward NPFF2 receptors and augmented differences among agonists. In low sodium conditions, whereas the potencies of agonists correlated with their affinities for NPFF1 receptors, NPFF2 receptors exhibited an extraordinary activity since all compounds tested displayed EC50 values of GTPγS binding lower than their K I values. Comparisons of functional values between NPFF1 and NPFF2 receptors revealed unexpected potent selective NPFF2 agonists especially for the PLRFamide and the VGRFamide sequences. By using blocker peptides, we also show that Gαi3 and Gαs are the main transducers of NPFF1 receptors while NPFF2 are probably coupled with Gαi2, Gαi3, Gαo and Gαs proteins. Our data indicate that NPPF1 and NPFF2 receptors are differently coupled to G proteins in CHO cells. [Copyright &y& Elsevier]