Binding of pleiotrophin to cell surface nucleolin mediates prostate cancer cell adhesion to osteoblasts.

Pleiotrophin (PTN) is a growth factor that appears to play an important role in prostate cancer growth and angiogenesis. We have previously shown that decreased PTN expression in human prostate cancer PC3 cells leads to decreased adhesion of prostate cancer cells to osteoblasts, suggesting that PTN mediates this interaction. In the current work, using peptides that correspond to different regions of the PTN protein, we identified that a domain responsible for the adhesion of prostate cancer cells to osteoblasts corresponds to amino acids 16-24 of the mature PTN protein. Given that a synthetic PTN16-24 peptide which disturbs the interaction of PTN with nucleolin (NCL) was found to inhibit prostate cancer cells' adhesion to osteoblasts, it seems that NCL mediates the cellular interactions involved in the adhesion process. Two pseudopeptides that bind to cell surface NCL and an anti-NCL antibody also decrease prostate cancer cell adhesion to osteoblasts to the same degree as PTN16-24, further supporting the involvement of cell surface NCL in this interaction. Collectively, our data suggest that NCL on the cell surface of osteoblasts may mediate adhesion of prostate cancer cells through PTN and identify peptides that could be exploited therapeutically to target this component of prostate cancer bone metastases.

The Use of Essential Oil and Hydrosol Extracted from Cuminum cyminum Seeds for the Control of Meloidogyne incognita and Meloidogyne javanica.

The essential oil (EO) and hydrosol (HL) isolated from Cuminum cyminum (cumin) seeds were evaluated against the root-knot nematodes Meloidogyne incognita and M. javanica. The efficacy of extracts on the motility, hatching, and survival in soil of second-stage juveniles (J2s), and the activity on egg differentiation were tested. All J2s were paralyzed after immersion in the EO at 62.5 µL/L concentration for 96 h. Encouraging results were recorded using HL equal to or higher than 10% concentration for both Meloidogyne species tested. More than 70% paralyzed J2s were recorded after immersion for 48 h, while the percentage was increased to higher than 90% after 96 h of immersion. A clear effect on egg differentiation was observed after immersion in EO or HL. A significant decrease in egg differentiation was revealed at even low concentrations of EO while an evident decrease in egg differentiation was recorded after immersion of eggs in 50% HL dilution. Decreased hatching of M. incognita and M. javanica J2s was observed with the increase in concentration. The lowest numbers of hatched J2s were recorded when EO was used at 1000 and 2000 µL/L concentrations. A constant reduction in root-knot nematode J2 hatching was observed upon increasing the concentration of HL from 5% to 50%. The EO of C. cyminum is characterized by the presence of γ-terpinene-7-al (34.95%), cumin aldehydes (26.48), and α-terpinene-7-al (12.77%). The above constituents were observed in HL following the same order as that observed in EO. The components γ-terpinene (11.09%) and ο-cymene (6.56%) were also recorded in EO while they were absent in HL.

Discovery of a stable tripeptide targeting the N-domain of CRF1 receptor.

The corticotropin-releasing factor (CRF) and its CRF1 receptor (CRF1R) play a central role in the maintenance of homeostasis. Malfunctioning of the CRF/CRF1R unit is associated with several disorders, such as anxiety and depression. Non-peptide CRF1R-selective antagonists have been shown to exert anxiolytic and antidepressant effects on experimental animals. However, none of them is in clinical use today because of several side effects, thus demonstrating the need for the development of other more suitable CRF1R antagonists. In an effort to develop novel CRF1R antagonists we designed, synthesized and chemically characterized two tripeptide analogues of CRF, namely (R)-LMI and (S)-LMI, having their Leu either in R (or D) or in S (or L) configuration, respectively. Their design was based on the crystal structure of the N-extracellular domain (N-domain) of CRF1R/CRF complex, using a relevant array of computational methods. Experimental evaluation of the stability of synthetic peptides in human plasma has revealed that (R)-LMI is proteolytically more stable than (S)-LMI. Based on this finding, (R)-LMI was selected for pharmacological characterization. We have found that (R)-LMI is a CRF antagonist, inhibiting (1) the CRF-stimulated accumulation of cAMP in HEK 293 cells expressing the CRF1R, (2) the production of interleukins by adipocytes and (3) the proliferation rate of RAW 264.7 cells. (R)-LMI likely blocked agonist actions by interacting with the N-domain of CRF1R as suggested by data using a constitutively active chimera of CRF1R. We propose that (R)-LMI can be used as an optimal lead compound in the rational design of novel CRF antagonists.

Nogo receptor complex expression dynamics in the inflammatory foci of central nervous system experimental autoimmune demyelination.

BACKGROUND: Nogo-A and its putative receptor NgR are considered to be among the inhibitors of axonal regeneration in the CNS. However, few studies so far have addressed the issue of local NgR complex multilateral localization within inflammation in an MS mouse model of autoimmune demyelination. METHODS: Chronic experimental autoimmune encephalomyelitis (EAE) was induced in C57BL/6 mice. Analyses were performed on acute (days 18-22) and chronic (day 50) time points and compared to controls. The temporal and spatial expression of the Nogo receptor complex (NgR and coreceptors) was studied at the spinal cord using epifluorescent and confocal microscopy or real-time PCR. Data are expressed as cells/mm2, as mean % ± SEM, or as arbitrary units of integrated density. RESULTS: Animals developed a moderate to severe EAE without mortality, followed by a progressive, chronic clinical course. NgR complex spatial expression varied during the main time points of EAE. NgR with coreceptors LINGO-1 and TROY was increased in the spinal cord in the acute phase whereas LINGO-1 and p75 signal seemed to be dominant in the chronic phase, respectively. NgR was detected on gray matter NeuN+ neurons of the spinal cord, within the white matter inflammatory foci (14.2 ± 4.3 % NgR+ inflammatory cells), and found to be colocalized with GAP-43+ axonal growth cones while no ß-TubIII+, SMI-32+, or APP+ axons were found as NgR+. Among the NgR+ inflammatory cells, 75.6 ± 9.0 % were microglial/macrophages (lectin+), 49.6 ± 14.2 % expressed CD68 (phagocytic ED1+ cells), and no cells were Mac-3+. Of these macrophages/monocytes, only Arginase-1+/NgR+ but not iNOS+/NgR+ were present in lesions both in acute and chronic phases. CONCLUSIONS: Our data describe in detail the expression of the Nogo receptor complex within the autoimmune inflammatory foci and suggest a possible immune action for NgR apart from the established inhibitory one on axonal growth. Its expression by inflammatory macrophages/monocytes could signify a possible role of these cells on axonal guidance and clearance of the lesioned area during inflammatory demyelination.

Alanine scanning mutagenesis of the second extracellular loop of type 1 corticotropin-releasing factor receptor revealed residues critical for peptide binding.

Upon binding of the corticotropin-releasing factor (CRF) analog sauvagine to the type 1 CRF receptor (CRF(1)), the amino-terminal portion of the peptide has been shown to lie near Lys257 in the receptor's second extracellular loop (EL2). To test the hypothesis that EL2 residues play a role in the binding of sauvagine to CRF(1) we carried out an alanine-scanning mutagenesis study to determine the functional role of EL2 residues (Leu251 to Val266). Only the W259A, F260A, and W259A/F260A mutations reduced the binding affinity and potency of sauvagine. In contrast, these mutations did not seem to significantly alter the overall receptor conformation, in that they left unchanged the affinities of the ligands astressin and antalarmin that have been suggested to bind to different regions of CRF(1). The W259A, F260A, and W259A/F260A mutations also decreased the affinity of the endogenous ligand, CRF, implying that these residues may play a common important role in the binding of different peptides belonging to CRF family. Parallel amino acid deletions of the two peptides produced ligands with various affinities for wild-type CRF(1) compared with the W259A, F260A, and W259A/F260A mutants, supporting the interaction between the amino-terminal residues 8 to 10 of sauvagine and the corresponding region in CRF with EL2 of CRF(1). This is the first time that a specific region of CRF(1) has been implicated in detailed interactions between the receptor and the amino-terminal portion of peptides belonging to the CRF family.

In vivo evaluation and in vitro metabolism of leuprolide in mice--mass spectrometry-based biomarker measurement for efficacy and toxicity.

The study of pharmacologically active peptides is central for the understanding of cancer and the development of novel therapeutic approaches. In this context, both qualitative and quantitative determination of bioactive peptides in biological fluids/tissues and their effect on endogenous factors (e.g. hormones) are of great importance. A mass spectrometry-based approach was developed and applied towards the measurement of leuprolide, a peptide drug for the treatment of prostate cancer, in mouse plasma. High-pressure liquid chromatography coupled to a hybrid quadrupole linear ion trap (QqLIT) mass spectrometer, a platform that combines the benefits of triple QqLIT instruments, was employed for the study. Using the described methodology, we established that picomolar concentrations of leuprolide could be measured in mouse plasma (limit of quantification of 0.1 ng/ml). In order to optimize pharmacokinetic properties of analogs of leuprolide, a facile in vivo mouse model was developed and leuprolide concentrations were determined in mouse plasma following intraperitoneal administration. In the same animal model, we demonstrated the versatility of the described MS-based approach by the determination of plasma concentrations of testosterone, an established biomarker for the treatment of prostate cancer. Following dosing with leuprolide, circulating testosterone was increased significantly in comparison to vehicle-treated mice. Finally, in vitro metabolism of leuprolide was evaluated by incubation of leuprolide with mouse kidney membranes, followed by identification of major metabolites by MS. Such studies provide the framework for future evaluation of novel leuprolide analogs with potential therapeutic advantages.