New Conjugates of Hyaluronic Acid with γ-Cyclodextrin as Sorafenib Carrier in Cancer Cells.

In recent years, nanoparticles based on cyclodextrins have been widely investigated, mainly for drug delivery. In this work, we synthesized nanoparticles with a hyaluronic acid backbone (11 kDa and 45 kDa) functionalized with γ-cyclodextrins. We tested sorafenib in the presence of the new hyaluronan-cyclodextrin conjugates in A2780 (ovarian cancer), SK-HeP-1 (adenocarcinoma) and MDA-MB-453 (breast cancer) cell lines. We found that hyaluronan-cyclodextrin conjugates improve the antiproliferative activity of sorafenib. Remarkably, the system based on the 11 kDa hyaluronan conjugate was the most effective and, in the MDA-MB-453 cell line, significantly reduced the IC50 value of sorafenib cells by about 75 %.

Aß and Tau Interact with Metal Ions, Lipid Membranes and Peptide-Based Amyloid Inhibitors: Are These Common Features Relevant in Alzheimer's Disease?

In the last two decades, the amyloid hypothesis, i.e., the abnormal accumulation of toxic Aß assemblies in the brain, has been considered the mainstream concept sustaining research in Alzheimer's Disease (AD). However, the course of cognitive decline and AD development better correlates with tau accumulation rather than amyloid peptide deposition. Moreover, all clinical trials of amyloid-targeting drug candidates have been unsuccessful, implicitly suggesting that the amyloid hypothesis needs significant amendments. Accumulating evidence supports the existence of a series of potentially dangerous relationships between Aß oligomeric species and tau protein in AD. However, the molecular determinants underlying pathogenic Aß/tau cross interactions are not fully understood. Here, we discuss the common features of Aß and tau molecules, with special emphasis on: (i) the critical role played by metal dyshomeostasis in promoting both Aß and tau aggregation and oxidative stress, in AD; (ii) the effects of lipid membranes on Aß and tau (co)-aggregation at the membrane interface; (iii) the potential of small peptide-based inhibitors of Aß and tau misfolding as therapeutic tools in AD. Although the molecular mechanism underlying the direct Aß/tau interaction remains largely unknown, the arguments discussed in this review may help reinforcing the current view of a synergistic Aß/tau molecular crosstalk in AD and stimulate further research to mechanism elucidation and next-generation AD therapeutics.

Cyclodextrin Polymers as Delivery Systems for Targeted Anti-Cancer Chemotherapy.

In the few last years, nanosystems have emerged as a potential therapeutic approach to improve the efficacy and selectivity of many drugs. Cyclodextrins (CyDs) and their nanoparticles have been widely investigated as drug delivery systems. The covalent functionalization of CyD polymer nanoparticles with targeting molecules can improve the therapeutic potential of this family of nanosystems. In this study, we investigated cross-linked γ- and ß-cyclodextrin polymers as carriers for doxorubicin (ox) and oxaliplatin (Oxa). We also functionalized γ-CyD polymer bearing COOH functionalities with arginine-glycine-aspartic or arginine moieties for targeting the integrin receptors of cancer cells. We tested the Dox and Oxa anti-proliferative activity in the presence of the precursor polymer with COOH functionalities and its derivatives in A549 (lung, carcinoma) and HepG2 (liver, carcinoma) cell lines. We found that CyD polymers can significantly improve the antiproliferative activity of Dox in HepG2 cell lines only, whereas the cytotoxic activity of Oxa resulted as enhanced in both cell lines. The peptide or amino acid functionalized CyD polymers, loaded with Dox, did not show any additional effect compared to the precursor polymer. Finally, studies of Dox uptake showed that the higher antiproliferative activity of complexes correlates with the higher accumulation of Dox inside the cells. The results show that CyD polymers could be used as carriers for repositioning classical anticancer drugs such as Dox or Oxa to increase their antitumor activity.

Novel Peptide-Calix[4]arene Conjugate Inhibits Aß Aggregation and Rescues Neurons from Aß's Oligomers Cytotoxicity In Vitro.

Alzheimer's disease (AD) is a progressive neurodegenerative condition affecting people in the elderly. Targeting aggregation of ß-amyloid peptides (Aß) is considered a promising approach for the therapeutic treatment of the disease. Peptide based inhibitors of ß-amyloid fibrillation are emerging as safe drug candidates as well as interesting compounds for early diagnosis of AD. Peptide conjugation via covalent bond with functional moieties enables the resultant hybrid system to acquire desired functions. Here we report the synthesis, the structural characterization, and the Aß42 interaction of a p-amino-calix[4]arene derivative bearing a GPGKLVFF peptide pendant at the lower rim. We demonstrate that the p-amino-calix[4]arene-GPGKLVFF conjugate alters the Aß42 aggregation pathways by preventing Aß42's conformational transition from random coil to ß-sheet with concomitant changes of the aggregation kinetic profile as evidenced by circular dichroism (CD), thioflavin T (ThT), and dynamic light scattering (DLS) measurements, respectively. High resolution mass spectrometry (HR-MS) confirmed a direct interaction of the p-amino-calix[4]arene-GPGKLVFF conjugate with Aß42 monomer which provided insight into a possible working mechanism, whereas the alteration of the Aß42's fibrillary architecture, by the calix-peptide conjugate, was further validated by atomic force microscopy (AFM) imaging. Finally, the herein proposed compound was shown to be effective against Aß42 oligomers' toxicity in differentiated neuroblastoma cells, SH-SY5Y.

Tau/Aß chimera peptides: A Thioflavin-T and MALDI-TOF study of Aß amyloidosis in the presence of Cu(II) or Zn(II) ions and total lipid brain extract (TLBE) vesicles.

Currently, Alzheimer's Disease (AD) is a complex neurodegenerative condition, with limited therapeutic options. Several factors, like Amyloid ß (Aß) aggregation, tau protein hyperphosphorylation, bio-metals dyshomeostasis and oxidative stress contribute to AD pathogenesis. These pathogenic processes might occur in the aqueous phase but also on neuronal membranes. Thus, investigating the connection between Aß and biomembranes, becomes important for unveiling the molecular mechanism underlying Aß amyloidosis as a critical event in AD pathology. In this work, the interaction of two peptides, made up with hybrid sequences from Tau protein 9-16 (EVMEDHAG) or 26-33 (QGGYTMHQ) N-terminal domain and Aß16-20 (KLVFF) hydrophobic region, with full length Aß40 or Aß42 peptides is reported. The studied "chimera" peptides Ac-EVMEDHAGKLVFF-NH2 (τ9-16-KL) and Ac-QGGYTMHQKLVFF-NH2 (τ26-33-KL) are endowed with Aß recognition and metal ion interaction capabilities provided by the tau or Aß sequences, respectively. These peptides were characterized in previous study along with their metal dependent interaction and amyloidogenesis, either in the presence or absence of metal ion and artificial membranes made up with Total Lipid Brain Extract (TLBE) components, (Sciacca et al., 2020). In the present paper, the ability of the two peptides to inhibit Aß aggregation is studied using composite experimental conditions including aqueous solution, the presence of metal ions (Cu or Zn), the presence of lipid vesicles mimicking neuronal membranes as well as the co-presence of metals and TLBE artificial membranes. We used Thioflavine-T (ThT) fluorescence or MALDI-TOF spectrometry analysis of Aß limited proteolysis to respectively monitor the Aß aggregation kinetic or validation of the Aß interacting regions. We demonstrate that τ9-16-KL and τ26-33-KL peptides differently affect Aß aggregation kinetics, with the tau sequence playing a crucial role. The results are discussed in terms of chimera's peptides hydrophobicity and electrostatic driven interactions at the aqueous/membrane interface.

Comparison of Centella with Flavonoids for Treatment of Symptoms in Hemorrhoidal Disease and After Surgical Intervention: A Randomized Clinical Trial.

Phlebotonics' effects were evaluated to reduce time-to-stop bleeding and anal irritation in 130 patients who complained of hemorrhoidal disease (HD); bleeding and pain after hemorrhoidectomy (31 patients) and hemorrhoidal thrombosis (34 patients) in the short time. Sixty patients were randomized to receive the routine treatment (both conservative and surgical) (control Group C). The treated group (both conservative and surgical) was divided into two subgroups: one treated with flavonoids (Group A, n = 73), the other with Centella (Group B, n = 66). Time-to-stop bleeding was checked at baseline and checkups (0 up to day 42). Healing was estimated with Kaplan-Meier method, the Kruskal-Wallis test estimated changes in the VAS scores. The HD median time-to-stop bleeding was 2 weeks for Groups A and B; 3 weeks for Group C. VAS scores comparison among Groups (irritation): A vs C, p = 0.007; B vs C, p = 0.041; and A vs B, p = 0.782 resulted respectively. As for operated hemorrhoids, the time-to-stop bleeding was 3 and 4 weeks in Groups A and B and 5 in Group C. Histopathology showed an association between flavonoids and piles' fibrosis (p = 0.008). Phlebotonics in HD, as well as after surgery, showed significant beneficial effects. Flavonoids are the most effective phlebotonics against bleeding and anal irritation.

The ability of the NiSOD binding loop to chelate zinc(ii): the role of the terminal amino group in the enzymatic functions.

Equilibrium and detailed spectroscopic characterization of zinc(ii) complexes with NiSOD binding loop and their related model fragments are reported in the whole investigated pH-range. The zinc(ii) complexes of L1 (HCDLPCGVY-NH2), L2 (Ac-HCDLPCGVY-NH2) and L3 (HCDLACGVY-NH2) and the nickel(ii) and zinc(ii) complexes of L4 (HCDLPCG-NH2) were studied by pH-potentiometric and several spectroscopic methods. The results indicated that the macrochelate coordinated zinc(ii) complexes are dominant in a whole pH-range and the side chain donors of the peptides are involved in the metal binding. Therefore, the deprotonation and coordination of the peptide backbone occur only in a strongly alkaline solution. The acetylation of the peptide amino terminus (L2) significantly enhances the zinc(ii) binding ability compared to the corresponding nickel(ii) complexes. L2 complexes of zinc(ii) are 2 or 3 orders of magnitude more stable than the corresponding nickel(ii) complexes. This effect clearly shows the crucial role of the terminal amino group in the nickel binding for the NiSOD enzyme.

Cyclodextrin polymers decorated with RGD peptide as delivery systems for targeted anti-cancer chemotherapy.

Polymeric cyclodextrin-based nanoparticles are currently undergoing clinical trials as nanotherapeutics. Using a non-covalent approach, we decorated two cross-linked cyclodextrin polymers of different molecular weights with an RGD peptide derivative to construct a novel carrier for the targeted delivery of doxorubicin. RGD is the binding sequence for the integrin receptor family that is highly expressed in tumour tissues. The assembled host-guest systems were investigated using NMR and DLS techniques. We found that, in comparison with free doxorubicin or the binary complex doxorubicin/cyclodextrin polymer, the RGD units decorating the cyclodextrin-based nanosystems improved the selectivity and cytotoxicity of the complexed doxorubicin towards cultured human tumour cell lines. Our results suggest that the nanocarriers under study may contribute to the development of new platforms for cancer therapy.

New comprehensive studies of a gold(III) Dithiocarbamate complex with proven anticancer properties: Aqueous dissolution with cyclodextrins, pharmacokinetics and upstream inhibition of the ubiquitin-proteasome pathway.

The gold(III)-dithiocarbamate complex AuL12 (dibromo [ethyl-N-(dithiocarboxy-kS,kS')-N-methylglycinate] gold(III)), is endowed with promising in vitro/in vivo antitumor activity and toxicological profile. Here, we report our recent strategies to improve its water solubility and stability under physiological conditions along with our efforts for unravelling its tangled mechanism of action. We used three types of α-cyclodextrins (CDs), namely ß-CD, Me-ß-CD and HP-ß-CD to prepare aqueous solutions of AuL12. The ability of these natural oligosaccharide carriers to enhance water solubility of hydrophobic compounds, allowed drug stability of AuL12 to be investigated. Moreover, pharmacokinetic experiments were first carried out for a gold(III) coordination compound, after i.v. injection of the nanoformulation AuL12/HP-ß-CD to female mice. The gold content in the blood samples was detected at scheduled times by AAS (atomic absorption spectrometry) analysis, highlighting a fast biodistribution with a tß1/2 of few minutes and a slow escretion (tα1/2 of 14.3 h). The in vitro cytotoxic activity of AuL12 was compared with the AuL12/HP-ß-CD mixture against a panel of three human tumor cell lines (i.e., HeLa, KB and MCF7). Concerning the mechanism of action, we previously reported the proteasome-inhibitory activity of some our gold(III)-based compounds. In this work, we moved from the proteasome target to upstream of the important ubiquitin-proteasome pathway, testing the effects of AuL12 on the polyubiquitination reactions involving the Ub-activating (E1) and -conjugating (E2) enzymes.

A rare cause of acute gastrointestinal hemorrhage: ileal lipoma Case report.

Primary small bowel tumors account for 1-6% of all Gastrointestinal (G.I.) tract malignancies. Among these pedunculated lipomas are unusual. We report a case of a 66-year-old male with a history of G.I. hemorrhage and acute anemia, negative upper and lower endoscopies and a pedunculated lipoma in distal ileum, revealed by CT enterography. The patient was successfully treated by open surgery. Patients with G.I. hemorrhage and negative upper and lower endoscopies need an accurate evaluation of small bowel. Multislice CT enterography or Magnetic Resonance enteroclysis/ enterography represent the fastest and more accurate tools to obtain an exhaustive evaluation of small bowel. In case of small bowel tumors this diagnostic procedures can show site and stage and can even suggest histological type of such neoplasms, with a significant impact in the surgical planning, avoiding time consuming surgical exploration. In this patient multislice TC enterography allowed a correct diagnosis of benign lipoma due to its radiological density and absence of infiltration of the intestinal wall and surrounding tissues. KEY WORDS: CT enterography, Gastrointestinal hemorrhage, Lipoma, Small bowel.

Early rectal cancer: a choice between local excision and transabdominal resection. A review of the literature and current guidelines.

INTRODUCTION: Indication for Local Excision (LE) or Trans Abdominal Resections with Total Mesorectal Excision (TAR) in Early Rectal Cancer (ERC) are still controversial. MATERIAL OF STUDY: We reviewed meta-analyses, scientific societies guidelines, randomized and controlled clinical trials from 1999 to 2016 for a total of 146,231 patients. We included in our analysis the accuracy of different tools of investigation, the reliability of the endoscopic biopsies and compared the results of the various LE and TAR. RESULTS: The Endo Rectal Ultra Sound (ERUS) is the most accurate technique for the preoperative staging with an 18% of understaging and a 17.3% of overstaging. Endoscopic biopsies do not provide reliable data on unfavorable histopathological features in a significant percentage of cases. The Transanal Excision Microsurgery (TEM) is the best technique among LE but with worse overall results than TAR in terms of R0, local recurrence and overall 5-years survival in T2 cancers. DISCUSSION: The ERUS is the diagnostic technique most appropriate in the preoperative staging of the ERC; the employment of Magnetic Resonance Imaging (MRI) has to be limited to uncertain T2 patients. The ERUS shows significant understaging rate which expose to inadequate treatment, particularly in T2 patients. Endoscopic biopsies risk to disregard unfavorable histological features, resulting in inadequate therapeutic indications to LE. The use of TAR guarantees overall better results than the use of LE on T2 and T1 with unfavorable histological findings. CONCLUSIONS: The TAR still shows best results in the ERC treatment especially in T2 and T1 with unfavorable histological findings. KEY WORDS: Early Rectal Cancer (ERC), Local Excision (LE), Trans Abdominal Resection with Total Mesorectal Excision (TAR-TME).

Copper(II) ions affect the gating dynamics of the 20S proteasome: a molecular and in cell study.

Due to their altered metabolism cancer cells are more sensitive to proteasome inhibition or changes of copper levels than normal cells. Thus, the development of copper complexes endowed with proteasome inhibition features has emerged as a promising anticancer strategy. However, limited information is available about the exact mechanism by which copper inhibits proteasome. Here we show that Cu(II) ions simultaneously inhibit the three peptidase activities of isolated 20S proteasomes with potencies (IC50) in the micromolar range. Cu(II) ions, in cell-free conditions, neither catalyze red-ox reactions nor disrupt the assembly of the 20S proteasome but, rather, promote conformational changes associated to impaired channel gating. Notably, HeLa cells grown in a Cu(II)-supplemented medium exhibit decreased proteasome activity. This effect, however, was attenuated in the presence of an antioxidant. Our results suggest that if, on one hand, Cu(II)-inhibited 20S activities may be associated to conformational changes that favor the closed state of the core particle, on the other hand the complex effect induced by Cu(II) ions in cancer cells is the result of several concurring events including ROS-mediated proteasome flooding, and disassembly of the 26S proteasome into its 20S and 19S components.

Influence of the N-terminus acetylation of Semax, a synthetic analog of ACTH(4-10), on copper(II) and zinc(II) coordination and biological properties.

Semax is a heptapeptide (Met-Glu-His-Phe-Pro-Gly-Pro) that encompasses the sequence 4-7 of N-terminal domain of the adrenocorticotropic hormone and a C-terminal Pro-Gly-Pro tripeptide. N-terminal amino group acetylation (Ac-Semax) modulates the chemical and biological properties of parental peptide, modifying the ability of Semax to form complex species with Cu(II) ion. At physiological pH, the main complex species formed by Ac-Semax, [CuLH-2]2-, consists in a distorted CuN3O chromophore with a weak apical interaction of the methionine sulphur. Such a complex differs from the Cu(II)-Semax complex system, which exhibits a CuN4 chromophore. The reduced ligand field affects the [CuLH-2]2- formal redox potential, which is more positive than that of Cu(II)-Semax corresponding species. In the amino-free form, the resulting complex species is redox-stable and unreactive against ascorbic acid, unlike the acetylated form. Semax acetylation did not protect from Cu(II) induced toxicity on a SH-SY5Y neuroblastoma cell line, thus demonstrating the crucial role played by the free NH2 terminus in the cell protection. Since several brain diseases are associated either to Cu(II) or Zn(II) dyshomeostasis, here we characterized also the complex species formed by Zn(II) with Semax and Ac-Semax. Both peptides were able to form Zn(II) complex species with comparable strength. Confocal microscopy imaging confirmed that peptide group acetylation does not affect the Zn(II) influx in neuroblastoma cells. Moreover, a punctuate distribution of Zn(II) within the cells suggests a preferred subcellular localization that might explain the zinc toxic effect. A future perspective can be the use of Ac-Semax as ionophore in antibody drug conjugates to produce a dysmetallostasis in tumor cells.

Tips to facilitate a preoperative diagnosis of adrenal ganglioneuroma. Report of a challenging experience and review of the literature.

AIM: Ganglioneuroma (GN) is the most uncommon and the most benign tumor among neuroblastic neoplasms, and in 29.7% of cases it finds in an adrenal gland. Usually asymptomatic, this tumor is detected incidentally in the majority of cases. It is generally challenging to obtain a precise diagnosis of adrenal ganglioneuroma (AGN) before surgery. Misdiagnosis rate of AGN on CT and MRI is 64.7% and clinicians and surgeons are often lacking in knowledge of this rare disease. For this reason, we pointed out the clinical, biochemical, radiologic and pathologic features of AGN in an our experience, with the aim to find out if there are some features able to facilitate a preoperative diagnosis. The present article also includes a review of the relevant literature in order to compare laparoscopic versus open adrenalectomy. CASE REPORT: Right AGN in a 42-year-old woman, in whom preoperative diagnosis was very difficult and only histopathological studies of the surgical specimen established the exact diagnosis. The patient underwent bilateral subcostal laparotomy for a large mass (sized measuring 14.5 x 11.6 x 6.5 cm.) and a right adrenalectomy was performed. Postoperative recovery was uneventful and the patient, at 12-months follow-up, is disease-free and in good health. DISCUSSION / CONCLUSIONS: Authors stress the importance of interdisciplinary collaboration between surgeons, radiologists and endocrinologists to optimize clinical management and surgical indications. Careful evaluation by endocrine examinations and multiple imaging procedures are necessary to provide a differential diagnosis. Surgeons should consider a diagnosis of AGN in case of: 1) an adrenal incidentaloma; 2) a nonfunctioning tumor with no elevated hormonal secretions, in which compressive symptoms may occur; 3) a homogeneous, encapsulated mass, with well-defined edges, without invasion of nearby structures (no vascular involvement), with presence of calcifications and nonenhanced attenuation of <40 HU on CT; 4) a homogeneous hypointense adrenal mass on T1-weighted MRI, a heterogeneous hyperintense mass on T2-weighted MRI and a poor delayed enhancement on dynamic MRI; a SUV level on PET less than 3.0. Nevertheless, diagnosis of AGN can be extremely challenging and can only be achieved by means of histology. Treatment is complete surgical resection without the need for chemotherapy or radiotherapy. Laparoscopic adrenalectomy is contraindicated in the presence of local infiltration or tumor greater than 12 cm. Even if AGN has an excellent prognosis and recurrences are rare after surgical resections, long-term follow-up is recommended. KEY WORDS: Adrenal gland, Adrenal ganglioneuroma, Laparoscopic adrenalectomy, Open adrenalectomy.

New Insight in Copper-Ion Binding to Human Islet Amyloid: The Contribution of Metal-Complex Speciation To Reveal the Polypeptide Toxicity.

Type-2 diabetes (T2D) is considered to be a potential threat on a global level. Recently, T2D has been listed as a misfolding disease, such as Alzheimer's and Parkinson's diseases. Human islet amyloid polypeptide (hIAPP) is a molecule cosecreted in pancreatic ß cells and represents the main constituent of an aggregated amyloid found in individuals affected by T2D. The trace-element serum level is significantly influenced during the development of diabetes. In particular, the dys-homeostasis of Cu(2+) ions may adversely affect the course of the disease. Conflicting results have been reported on the protective role played by complex species formed by Cu(2+) ions with hIAPP or its peptide fragments in vitro. The histidine (His) residue at position 18 represents the main binding site for the metal ion, but contrasting results have been reported on other residues involved in metal-ion coordination, in particular those toward the N or C terminus. Sequences that encompass regions 17-29 and 14-22 were used to discriminate between the two models of the hIAPP coordination mode. Due to poor solubility in water, poly(ethylene glycol) (PEG) derivatives were synthesized. A peptide fragment that encompasses the 17-29 region of rat amylin (rIAPP) in which the arginine residue at position 18 was substituted by a histidine residue was also obtained to assess that the PEG moiety does not alter the peptide secondary structure. The complex species formed by Cu(2+) ions with Ac-PEG-hIAPP(17-29)-NH2 , Ac-rIAPP(17-29)R18H-NH2 , and Ac-PEG-hIAPP(14-22)-NH2 were studied by using potentiometric titrations coupled with spectroscopic methods (UV/Vis, circular dichroism, and EPR). The combined thermodynamic and spectroscopic approach allowed us to demonstrate that hIAPP is able to bind Cu(2+) ions starting from the His18 imidazole nitrogen atom toward the N-terminus domain. The stability constants of copper(II) complexes with Ac-PEG-hIAPP(14-22)-NH2 were used to simulate the different experimental conditions under which aggregate formation and oxidative stress of hIAPP has been reported. Speciation unveils: 1) the protective role played by increased amounts of Cu(2+) ions on the hIAPP fibrillary aggregation, 2) the effect of adventitious trace amounts of Cu(2+) ions present in phosphate-buffered saline (PBS), and 3) a reducing fluorogenic probe on H2 O2 production attributed to the polypeptide alone.

On the Environmental Factors Affecting the Structural and Cytotoxic Properties of IAPP Peptides.

Pancreatic islets in type 2 diabetes mellitus (T2DM) patients are characterized by reduced ß-cells mass and diffuse extracellular amyloidosis. Amyloid deposition involves the islet amyloid polypeptide (IAPP), a neuropancreatic hormone cosecreted with insulin by ß-cells. IAPP is physiologically involved in glucose homeostasis, but it may turn toxic to ß-cells owing to its tendency to misfold giving rise to oligomers and fibrils. The process by which the unfolded IAPP starts to self-assemble and the overall factors promoting this conversion are poorly understood. Other open questions are related to the nature of the IAPP toxic species and how exactly ß-cells die. Over the last decades, there has been growing consensus about the notion that early molecular assemblies, notably small hIAPP oligomers, are the culprit of ß-cells decline. Numerous environmental factors might affect the conformational, aggregation, and cytotoxic properties of IAPP. Herein we review recent progress in the field, focusing on the influences that membranes, pH, and metal ions may have on the conformational conversion and cytotoxicity of full-length IAPP as well as peptide fragments thereof. Current theories proposed for the mechanisms of toxicity will be also summarized together with an outline of the underlying molecular links between IAPP and amyloid beta (Aß) misfolding.

The N-Terminal Peptides of the Three Human Isoforms of the Mitochondrial Voltage-Dependent Anion Channel Have Different Helical Propensities.

The voltage-dependent anion channel (VDAC) is the main mitochondrial porin allowing the exchange of ions and metabolites between the cytosol and the mitochondrion. In addition, VDAC was found to actively interact with proteins playing a fundamental role in the regulation of apoptosis and being of central interest in cancer research. VDAC is a large transmembrane ß-barrel channel, whose N-terminal helical fragment adheres to the channel interior, partially closing the pore. This fragment is considered to play a key role in protein stability and function as well as in the interaction with apoptosis-related proteins. Three VDAC isoforms are differently expressed in higher eukaryotes, for which distinct and complementary roles are proposed. In this work, the folding propensity of their N-terminal fragments has been compared. By using multiple spectroscopic techniques, and complementing the experimental results with theoretical computer-assisted approaches, we have characterized their conformational equilibrium. Significant differences were found in the intrinsic helical propensity of the three peptides, decreasing in the following order: hVDAC2 > hVDAC3 > hVDAC1. In light of the models proposed in the literature to explain voltage gating, selectivity, and permeability, as well as interactions with functionally related proteins, our results suggest that the different chemicophysical properties of the N-terminal domain are possibly correlated to different functions for the three isoforms. The overall emerging picture is that a similar transmembrane water accessible conduit has been equipped with not identical domains, whose differences can modulate the functional roles of the three VDAC isoforms.

Cross-talk between the octarepeat domain and the fifth binding site of prion protein driven by the interaction of copper(II) with the N-terminus.

Prion diseases are a group of neurodegenerative diseases based on the conformational conversion of the normal form of the prion protein (PrP(C)) to the disease-related scrapie isoform (PrP(Sc)). Copper(II) coordination to PrP(C) has attracted considerable interest for almost 20 years, mainly due to the possibility that such an interaction would be an important event for the physiological function of PrP(C). In this work, we report the copper(II) coordination features of the peptide fragment Ac(PEG11)3PrP(60-114) [Ac = acetyl] as a model for the whole N-terminus of the PrP(C) metal-binding domain. We studied the complexation properties of the peptide by means of potentiometric, UV/Vis, circular dichroism and electrospray ionisation mass spectrometry techniques. The results revealed that the preferred histidyl binding sites largely depend on the pH and copper(II)/peptide ratio. Formation of macrochelate species occurs up to a 2:1 metal/peptide ratio in the physiological pH range and simultaneously involves the histidyl residues present both inside and outside the octarepeat domain. However, at increased copper(II)/peptide ratios amide-bound species form, especially within the octarepeat domain. On the contrary, at basic pH the amide-bound species predominate at any copper/peptide ratio and are formed preferably with the binding sites of His96 and His111, which is similar to the metal-binding-affinity order observed in our previous studies.

Semax, an ACTH4-10 peptide analog with high affinity for copper(II) ion and protective ability against metal induced cell toxicity.

Heptapeptide Semax, encompassing the sequence 4-7 of N-terminal domain of the adrenocorticotropic hormone (ACTH) and a C-terminal Pro-Gly-Pro tripeptide, belongs to a short regulatory peptides family. This compound has been found to affect learning processes and to exert marked neuroprotective activities on cognitive brain functions. Dys-homeostasis of metal ions is involved in several neurodegenerative disorders and growing evidences have showed that brain is a specialized organ able to concentrate metal ions. In this work, the metal binding ability and protective activity of Semax and its metal complexes were studied. The equilibrium study clearly demonstrated the presence of three complex species. Two minor species [CuL] and [CuLH-1]- co-exist together with the [CuLH-2]2- in the pH range from 3.6 to 5. From pH5 the [CuLH-2]2- species becomes predominant with the donor atoms around copper arranged in a 4N planar coordination mode. Noteworthy, a reduced copper induced cytotoxicity was observed in the presence of Semax by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay on a SHSY5Y neuroblastoma and RBE4 endothelial cell lines.

Choledochal cyst mimicking Mirizzi's syndrome: a case report.

Choledochal cysts are cystic dilatations of the intra or extra-hepatic biliary tract with an incidence of 1 case per 150.000 live births. Cysts usually are diagnosed in childhood, but diagnosis can be delayed until adulthood in the 20-50% of cases. Clinical manifestations comprise abdominal pain with biliary or pancreatic features. Mirizzi's syndrome is a late and rare complication, that occurs in 1% of patients with cholelithiasis due to extrinsic compression of the common bile duct by stones impacted either in the gallbladder or in the cystic duct. Clinical symptoms include extrahepatic obstructive jaundice, ascending cholangitis, or, in the later course, cholecystocholedocal fistula. For both pathologies the Endoscopic Retrograde Cholangio Pancreatography and the Magnetic Resonance Cholangio Pancreatography should lead to the diagnosis with a sensibility and a specificity up to 100%. We report the case of a 66 year old patient admitted to the Emergency Department of our hospital for jaundice and abdominal pain, whom both the endoscopic and radiologic examination showed a Mirizzi's syndrome but surgery revealed a type I choledocal cyst.