Pietro Campiglia

OBESTATIN PROMOTES SURVIVAL OF PANCREATIC β-CELLS AND HUMAN ISLETS AND INDUCES EXPRESSION OF GENES INVOLVED IN THE REGULATION OF β-CELL MASS AND FUNCTION

OBJECTIVE—Obestatin is a newly discovered peptide encoded by the ghrelin gene whose biological functions are poorly understood. We investigated obestatin effect on survival of β-cells and human pancreatic islets and the underlying signaling pathways. RESEARCH DESIGN AND METHODS—β-Cells and human islets were used to assess obestatin effect on cell proliferation, survival, apoptosis, intracellular signaling, and gene expression. RESULTS—Obestatin showed specific binding on HIT-T15 and INS-1E β-cells, bound to glucagon-like peptide-1 receptor (GLP-1R), and recognized ghrelin binding sites. Obestatin exerted proliferative, survival, and antiapoptotic effects under serum-deprived conditions and interferon-γ/tumor necrosis factor-α/interleukin-1β treatment, particularly at pharmacological concentrations. Ghrelin receptor antagonist [D-Lys3]-growth hormone releasing peptide-6 and anti-ghrelin antibody prevented obestatin-induced survival in β-cells and human islets. β-Cells and islet cells released obestatin, and addition of anti-obestatin antibody reduced their viability. Obestatin increased β-cell cAMP and activated extracellular signal–related kinase 1/2 (ERK1/2) and phosphatidylinositol 3-kinase (PI 3-kinase)/Akt; its antiapoptotic effect was blocked by inhibition of adenylyl cyclase/cAMP/protein kinase A (PKA), PI 3-kinase/Akt, and ERK1/2 signaling. Moreover, obestatin upregulated GLP-1R mRNA and insulin receptor substrate-2 (IRS-2) expression and phosphorylation. The GLP-1R antagonist exendin-(9-39) reduced obestatin effect on β-cell survival. In human islets, obestatin, whose immunoreactivity colocalized with that of ghrelin, promoted cell survival and blocked cytokine-induced apoptosis through cAMP increase and involvement of adenylyl cyclase/cAMP/PKA signaling. Moreover, obestatin 1) induced PI 3-kinase/Akt, ERK1/2, and also cAMP response element–binding protein phosphorylation; 2) stimulated insulin secretion and gene expression; and 3) upregulated GLP-1R, IRS-2, pancreatic and duodenal homeobox-1, and glucokinase mRNA. CONCLUSIONS—These results indicate that obestatin promotes β-cell and human islet cell survival and stimulates the expression of main regulatory β-cell genes, identifying a new role for this peptide within the endocrine pancreas.

The G protein coupled receptor kinase 2 plays an essential role in beta-adrenergic receptor-induced insulin resistance

AIMS Insulin (Ins) resistance (IRES) associates to increased cardiovascular risk as observed in metabolic syndrome. Chronic stimulation of beta-adrenergic receptors (betaAR) due to exaggerated sympathetic nervous system activity is involved in the pathogenesis of IRES. The cellular levels of G protein coupled receptor kinase 2 (GRK2) increase during chronic betaAR stimulation, leading to betaAR desensitization. We tested the hypothesis that GRK2 plays a role in betaAR-induced IRES. METHODS AND RESULTS We evaluated Ins-induced glucose uptake and signalling responses in vitro in cell overexpressing the beta(2)AR, the GRK2, or the catalytically dead mutant GRK2-DN. In a model of increased adrenergic activity, IRES and elevated cellular GRK2 levels, the spontaneously hypertensive rats (SHR) we performed the intravenous glucose tolerance test load. To inhibit GRK2, we synthesized a peptide based on the catalytical sequence of GRK2 conjugated with the antennapedia internalization sequence (Ant-124). Ins in human kidney embryonic (HEK-293) cells causes rapid accumulation of GRK2, tyrosine phosphorylation of Ins receptor substrate 1 (IRS1) and induces glucose uptake. In the same cell type, transgenic beta(2)AR overexpression causes GRK2 accumulation associated with significant deficit of IRS1 activation and glucose uptake by Ins. Similarly, transgenic GRK2 overexpression prevents Ins-induced tyrosine phosphorylation of IRS1 and glucose uptake, whereas GRK2-DN ameliorates glucose extraction. By immunoprecipitation, GRK2 binds IRS1 but not the Ins receptor in an Ins-dependent fashion, which is lost in HEK-GRK2 cells. Ant-124 improves Ins-induced glucose uptake in HEK-293 and HEK-GRK2 cells, but does not prevent GRK2/IRS1 interaction. In SHR, Ant-124 infusion for 30 days ameliorates IRES and IRS1 tyrosine phosphorylation. CONCLUSION Our results suggest that GRK2 mediates adrenergic IRES and that inhibition of GRK2 activity leads to increased Ins sensitivity both in cells and in animal model of IRES.

New arylthioindoles and related bioisosteres at the sulfur bridging group. 4. Synthesis, tubulin polymerization, cell growth inhibition, and molecular modeling studies.

New arylthioindoles along with the corresponding ketone and methylene compounds were potent tubulin assembly inhibitors. As growth inhibitors of MCF-7 cells, sulfur derivatives were superior or sometimes equivalent to the ketones, while methylene derivatives were substantially less effective. Esters 24, 27-29, 36, 39, and 41 showed approximately 50% of inhibition on human HeLa and HCT116/chr3 cells at 0.5 microM, and these compounds inhibited the growth of HEK, M14, and U937 cells with IC(50)s in the 78-220 nM range. While murine macrophage J744.1 cell growth was significantly less affected (20% at higher concentrations), four other nontransformed cell lines remained sensitive to these esters. The effect of drug treatment on cell morphology was examined by time-lapse microscopy. In a protocol set up to evaluate toxicity on the Saccharomyces cerevisiae BY4741 wild type strain, compounds 24 and 54 strongly reduced cell growth, and 29, 36, and 39 also showed significant inhibition.

Flavonoid Fraction of Bergamot Juice Reduces LPS-Induced Inflammatory Response through SIRT1-Mediated NF-κB Inhibition in THP-1 Monocytes

Plant polyphenols exert anti-inflammatory activity through both anti-oxidant effects and modulation of pivotal pro-inflammatory genes. Recently, Citrus bergamia has been studied as a natural source of bioactive molecules with antioxidant activity, but few studies have focused on molecular mechanisms underlying their potential beneficial effects. Several findings have suggested that polyphenols could influence cellular function by acting as activators of SIRT1, a nuclear histone deacetylase, involved in the inhibition of NF-κB signaling. On the basis of these observations we studied the anti-inflammatory effects produced by the flavonoid fraction of the bergamot juice (BJe) in a model of LPS-stimulated THP-1 cell line, focusing on SIRT1-mediated NF-κB inhibition. We demonstrated that BJe inhibited both gene expression and secretion of LPS-induced pro-inflammatory cytokines (IL-6, IL-1β, TNF-α) by a mechanism involving the inhibition of NF-κB activation. In addition, we showed that BJe treatment reversed the LPS-enhanced acetylation of p65 in THP-1 cells. Interestingly, increasing concentrations of Sirtinol were able to suppress the inhibitory effect of BJe via p65 acetylation, underscoring that NF-κB–mediated inflammatory cytokine production may be directly linked to SIRT1 activity. These results suggest that BJe may be useful for the development of alternative pharmacological strategies aimed at reducing the inflammatory process.

A Different Molecular Mechanism Underlying Antimicrobial and Hemolytic Actions of Temporins A and L

In this work, the naturally occurring antimicrobial peptides temporin A (TA) and L (TL) are studied by spectroscopic (CD and NMR) techniques and molecular dynamics simulation. We analyzed the interactions of TA and TL with sodium dodecyl sulfate (SDS) and dodecylphosphocholine (DPC) micelles, which mimic bacterial and mammalian membranes, respectively. In SDS, the peptides prefer a location at the micelle-water interface; in DPC, they prefer a location perpendicular to the micelle surface, with the N-terminus imbedded in the hydrophobic core. TL shows higher propensity, with respect to TA, in forming alpha-helical structures in both membrane mimetic systems and the highest propensity to penetrate the micelles. Hence, we have proposed a different molecular mechanism underlying the antimicrobial and hemolytic activities of the two peptides. We also designed new analogues of TA and TL and found interesting differences in their efficacy against microbial species and human erythrocytes.

Peptides from Royal Jelly: studies on the antimicrobial activity of jelleins, jelleins analogs and synergy with temporins

Peptides isolated from natural fonts are the object of several studies aimed at finding new molecules possessing antibacterial activity. We focused our studies on peptides originally isolated from the Royal Jelly, the jelleins and on some analogs having a UV reporter at the N‐ or C‐terminus. We found that jelleins are mainly active against gram‐positive bacteria; interestingly, they act in synergy with peptides belonging to the family of temporins such as temporin A and temporin B against Staphylococcus aureus A170 and Listeria monocytogenes. Copyright

Calmodulin-Dependent Kinase II Mediates Vascular Smooth Muscle Cell Proliferation and Is Potentiated by Extracellular Signal Regulated Kinase

Vascular smooth muscle cell (VSMC) proliferation contributes to vascular remodeling in atherosclerosis and hypertension. Calcium-dependent signaling through calcium/calmodulin-dependent kinase II (CaMKII) and ERK1/2 activation plays an important role in the regulation of VSMC proliferation by agents such as alpha-adrenergic receptor agonists. Nevertheless, how the CaMKII and ERK pathways interact in VSMCs has yet to be characterized. The aim of the present study was to clarify this interaction in response to alpha(1)-adrenergic receptor-mediated VSMC proliferation. We discovered that phenylephrine stimulation resulted in complex formation between CaMKII and ERK in a manner that facilitated phosphorylation of both protein kinases. To assess the effects of CaMKII/ERK association on VSMC proliferation, we inhibited endogenous CaMKII either pharmacologically or by adenoviral-mediated gene transfer of a kinase-inactive CaMKII mutant. Inhibition of CaMKII activation but not CaMKII autonomous activity significantly decreased formation of the CaMKII/ERK complex. On the contrary, the expression of constitutively active CaMKII enhanced VSMC growth and CaMKII/ERK association. In addressing the mechanism of this effect, we found that CaMKII could not directly phosphorylate ERK but instead enhanced Raf1 activation. By contrast, ERK interaction with CaMKII facilitated CaMKII phosphorylation and promoted its nuclear localization. Our results reveal a critical role for CaMKII in VSMC proliferation and imply that CaMKII facilitates assembly of the Raf/MEK/ERK complex and that ERK enhances CaMKII activation and influences its subcellular localization.

Online Comprehensive RPLC × RPLC with Mass Spectrometry Detection for the Analysis of Proteome Samples

LC-MS-based shotgun proteomics relies both on the power of the separation techniques and the sensitivity of detection methods. As a viable alternative to classical approaches in this field, we developed a fully automated, comprehensive 2D LC system, in which RPLC × RPLC was coupled to MS detection, for the first time, and applied for the analysis of tryptic digests obtained from α-casein and dephosphorylated α-casein. The use of a significantly different pH in the two dimensions allowed us to attain high peak capacity, despite the employment of novel identical stationary phases. Furthermore, such a combination addresses compatibility issues, thus allowing straightforward interfacing in online 2D LC configuration, as well as direct linkage to a mass spectrometer. A theoretical peak capacity of ca. 8500 was calculated for the setup, employing four serially coupled C18 columns in the first dimension (600 × 2.1 mm, 2.7 μm d.p.), operated under basic conditions, and 3 cm length of the same stationary phase (30 × 4.6 mm, 2.7 μm d.p. column), under acidic conditions, for fast second dimension analysis.

Structure-activity relationship, conformational and biological studies of temporin l analogues

Temporins are naturally occurring peptides with promising features, which could lead to the development of new drugs. Temporin-1Tl (TL) is the strongest antimicrobial peptide, but it is toxic on human erythrocytes and this fact makes the design of synthetic analogues with a higher therapeutic index vital.We studied the structure-activity relationships of a library of TL derivatives focusing on the correlation between the α-helix content of the peptides, the nature of their cationic residues, and their antibacterial/antiyeast/hemolytic activities. We found that the percentage of helicity of TL analogues is directly correlated to their hemolytic activity but not to their antimicrobial activity. In addition, we found that the nature of positively charged residues can affect the biological properties of TL without changing the peptides helicity. It is noteworthy that a single amino acid substitution can prevent the antimicrobial activity of TL, making it a lytic peptide presumably due to its self-association. Last, we identified a novel analogue with properties that make it an attractive topic for future research.

Human Glioblastoma Multiforme: p53 Reactivation by a Novel MDM2 Inhibitor

Cancer development and chemo-resistance are often due to impaired functioning of the p53 tumor suppressor through genetic mutation or sequestration by other proteins. In glioblastoma multiforme (GBM), p53 availability is frequently reduced because it binds to the Murine Double Minute-2 (MDM2) oncoprotein, which accumulates at high concentrations in tumor cells. The use of MDM2 inhibitors that interfere with the binding of p53 and MDM2 has become a valid approach to inhibit cell growth in a number of cancers; however little is known about the efficacy of these inhibitors in GBM. We report that a new small-molecule inhibitor of MDM2 with a spirooxoindolepyrrolidine core structure, named ISA27, effectively reactivated p53 function and inhibited human GBM cell growth in vitro by inducing cell cycle arrest and apoptosis. In immunoincompetent BALB/c nude mice bearing a human GBM xenograft, the administration of ISA27 in vivo activated p53, inhibited cell proliferation and induced apoptosis in tumor tissue. Significantly, ISA27 was non-toxic in an in vitro normal human cell model and an in vivo mouse model. ISA27 administration in combination with temozolomide (TMZ) produced a synergistic inhibitory effect on GBM cell viability in vitro, suggesting the possibility of lowering the dose of TMZ used in the treatment of GBM. In conclusion, our data show that ISA27 releases the powerful antitumor capacities of p53 in GBM cells. The use of this MDM2 inhibitor could become a novel therapy for the treatment of GBM patients.