An Italian pilot study of a psycho-social intervention to support family caregivers' engagement in taking care of patients with complex care needs: the Engage-in-Caring project.

BACKGROUND: The raising of disability and chronic illness burden among European population is calling for a new paradigm of care, focused on primary health care interventions. Engage-In-Caring is a novel multicomponent intervention clearly dedicated to improve family caregiver engagement in the care of patients with complex care needs, by supporting them to develop a stronger consciousness of their role, needs and skills. METHOD: Engage-In-Caring intervention's efficacy and feasibility have been evaluated through a single arm pre-post observational pilot study settled in Rome. A qualitative phase, consisting of literature analysis of caregivers' unmet needs and a final revision from an experts' group, led to the structuration of the intervention, following the Caregiver Health Engagement Model (CHE-Model). Afterwards, a quantitative phase allowed understanding the feasibility of the intervention through Kruskal-Wallis test on a sample of 47 caregivers. RESULTS: Results showed a reduction of the physical burden (Chi Squared = 6,483; p = .01) perceived by the caregivers and increase of the health literacy (Chi Squared = 3,560; p = .059) after the intervention. CONCLUSIONS: Feasibility tests on caregivers of patients with complex care needs are promising: this pilot study suggests a first effectiveness evidence, particularly concerning aspects related to burden perception and improvements in health literacy. Randomised controlled trials on larger samples are needed.

Neurotoxic properties of the anabolic androgenic steroids nandrolone and methandrostenolone in primary neuronal cultures.

Anabolic-androgenic steroid (AAS) abuse is associated with multiple neurobehavioral disturbances. The sites of action and the neurobiological sequels of AAS abuse are unclear at present. We investigated whether two different AASs, nandrolone and methandrostenolone, could affect neuronal survival in culture. The endogenous androgenic steroid testosterone was used for comparison. Both testosterone and nandrolone were neurotoxic at micromolar concentrations, and their effects were prevented by blockade of androgen receptors (ARs) with flutamide. Neuronal toxicity developed only over a 48-hr exposure to the steroids. The cell-impermeable analogues testosterone-BSA and nandrolone-BSA, which preferentially target membrane-associated ARs, were also neurotoxic in a time-dependent and flutamide-sensitive manner. Testosterone-BSA and nandrolone-BSA were more potent than their parent compounds, suggesting that membrane-associated ARs were the relevant sites for the neurotoxic actions of the steroids. Unlike testosterone and nandrolone, toxicity by methandrostenolone and methandrostenolone-BSA was insensitive to flutamide, but it was prevented by the glucocorticoid receptor (GR) antagonist RU-486. Methandrostenolone-BSA was more potent than the parent compound, suggesting that its toxicity relied on the preferential activation of putative membrane-associated GRs. Consistently with the evidence that membrane-associated GRs can mediate rapid effects, a brief challenge with methandrostenolone-BSA was able to promote neuronal toxicity. Activation of putative membrane steroid receptors by nontoxic (nanomolar) concentrations of either nandrolone-BSA or methandrostenolone-BSA became sufficient to increase neuronal susceptibility to the apoptotic stimulus provided by ß-amyloid (the main culprit of AD). We speculate that AAS abuse might facilitate the onset or progression of neurodegenerative diseases not usually linked to drug abuse.

The interaction of humic substances with the human prion protein fragment 90-231 affects its protease K resistance and cell internalization.

In this paper we analyzed the determinants and the structural effects of the interaction of human prion protein fragment 90-231 (HuPrP) with humic substances, (HS) including humic (HA) and fulvic (FA) acids, natural refractory organic polyanions widely diffused in soils and waters. We show that this interaction is mainly driven by non-specific electrostatic attraction involving regions situated within alpha-helix A and beta-sheet S1 of human PrP. FA binding to HuPrP altered its ability to acquire some PrPSc-like characteristics induced by the mild thermal denaturation of the peptide (1 h at 53 degrees C). In particular, in the presence of FA, HuPrP shows a reduced amount of beta-sheet content (as demonstrated by the reduced binding of thioflavin T), an increased sensitivity to protease K and an inhibition of the entering in the fibrillogenic pathway. FA/HuPrP interaction caused the aggregation of the peptide in unstructured macrocomplexes, as demonstrated by the altered electrophoretic migration in semi-denaturing detergent-agarose gel assay. Importantly, in the presence of FA the rate of internalization of HuPrP in human neuroblastoma cells was significantly reduced as compared to that of the beta-structured peptide. Therefore, HS inhibited the acquisition of PrP(Sc)-like structural properties that, in turn, are responsible for HuPrP intracellular accumulation and lead to neuronal death. Important implications of these data are that HuPrP-HS complexes, being unable to be internalized in living cells may represent a molecular mechanism for the reduced transmission of prion transmission from HS-rich soil also in the presence of contamination from infected animals.

Conformation dependent pro-apoptotic activity of the recombinant human prion protein fragment 90-231.

The transition of prion protein from a mainly alpha-structured isoform (PrPC) to a beta sheet-containing protein (PrPSc) represents a major pathogenetic mechanism in prion diseases. To study the role of PrP structural conformation in prion-dependent neurodegeneration, we analysed the neurotoxicity of PrP in alpha and beta conformations, using a recombinant protein encompassing amino acids 90-231 of the human PrP (hPrP90-231). Using controlled thermal denaturation (53 degrees C, 1h) we converted hPrP90-231 in a structural isoform displaying PrPSc-related characteristics: high beta sheet content, increased aggregability and a slight increase in the resistance to protease K. In virtue of these structural changes, hPrP90-231 powerfully affected the survival of SH-SY5Y cells, inducing a caspase-3 and p38- dependent apoptosis. Conversely, in the native alpha-helix-rich conformation, hPrP90-231 did not show significant cell toxicity. The relationship between the structural state of hPrP90-231 and its neurotoxicity was demonstrated, inducing the thermal denaturation of the peptide in the presence of Congo red that prevented both the transition of hPrP90-231 into a beta-rich isoform and the acquisition of toxic properties. In conclusion, we report that the toxicity of hPrP90-231 is dependent on its three-dimensional structure, as is supposed to occur for the pathogen PrP during TSE.

Identification of a conserved N-capping box important for the structural autonomy of the prion alpha 3-helix: the disease associated D202N mutation destabilizes the helical conformation.

Peptides corresponding to three alpha helices present in the C-terminal region of the human prion protein have been synthesized and their structural autonomy analyzed by circular dichroism (CD) and NMR spectroscopy. The results obtained indicate that the protein fragment corresponding to the alpha 3-helix, in contrast to alpha 1 and alpha 2 peptides, shows a complete structural autonomy. The chemical shifts values found for NH and CHalpha resonance of the isolated alpha 3 peptide, formed by 30 aminoacid residues, were markedly and surprisingly similar to the corresponding values of the alpha 3-helix in the protein. The structural autonomy of the alpha 3-helix is profoundly determined by the presence of the conserved capping box and, in part, by the ionic bond formed between Glu200 and Lys204. On the basis of these observations a novel PrP consensus pattern, centered on the alpha 3-helix region, has been defined. The data indicate that this autonomous and highly conserved region of the PrPc likely plays a critical role in folding and stability. This gives an explanation of why many of pathogenic mutations occur in this part of the molecule, sharing relevant effects on the overall protein conformation. In particular the D202N capping mutation almost completely destabilizes the isolated alpha 3 peptide. While it is well known that the D202N substitution is associated with a GSS disease, the possible structural basis of this fatal pathology has never been investigated. We propose that a lower alpha 3-helical propensity leading to a major destabilization of the PrPc molecule initiates the pathogenic process associated with D202N capping mutation.

The activation of the phosphotyrosine phosphatase eta (r-PTP eta) is responsible for the somatostatin inhibition of PC Cl3 thyroid cell proliferation.

The aim of this study was the characterization of the intracellular effectors of the antiproliferative activity of somatostatin in PC Cl3 thyroid cells. Somatostatin inhibited PC Cl3 cell proliferation through the activation of a membrane phosphotyrosine phosphatase. Conversely, PC Cl3 cells stably expressing the v-mos oncogene (PC mos) were completely insensitive to the somatostatin antiproliferative effects since somatostatin was unable to stimulate a phosphotyrosine phosphatase activity. In PC mos cells basal phosphotyrosine phosphatase activity was also reduced, suggesting that the expression of a specific phosphotyrosine phosphatase was impaired in these transformed cells. We suggested that this phosphotyrosine phosphatase could be r-PTP eta whose expression was abolished in the PC mos cells. To directly prove the involvement of r-PTP eta in somatostatin's effect, we stably transfected this phosphatase in PC mos cells. This new cell line (PC mos/PTP eta) recovered somatostatin's ability to inhibit cell proliferation, showing dose-dependence and time course similar to those observed in PC Cl3 cells. Conversely, the transfection of a catalytically inactive mutant of r-PTP eta did not restore the antiproliferative effects of somatostatin. PC mos/PTP eta cells showed a high basal phosphotyrosine phosphatase activity which, similarly to PC Cl3 cells, was further increased after somatostatin treatment. The specificity of the role of r-PTP eta in somatostatin receptor signal transduction was demonstrated by measuring its specific activity after somatostatin treatment in an immunocomplex assay. Somatostatin highly increased r-PTP eta activity in PCCl3 and PC mos/PTP eta (+300%, P < 0.01) but not in PCmos cells. Conversely, no differences in somatostatin-stimulated SHP-2 activity, (approximately +50%, P < 0.05), were observed among all the cell lines. The activation of r-PTP eta by somatostatin caused, acting downstream of MAPK kinase, an inhibition of insulin-induced ERK1/2 activation with the subsequent blockade of the phosphorylation, ubiquitination, and proteasome degradation of the cyclin-dependent kinase inhibitor p27(kip1). Ultimately, high levels of p27(kip1) lead to cell proliferation arrest. In conclusion, somatostatin inhibition of PC Cl3 cell proliferation requires the activation of r-PTP eta which, through the inhibition of MAPK activity, causes the stabilization of the cell cycle inhibitor p27(kip1).

Somatostatin inhibits tumor angiogenesis and growth via somatostatin receptor-3-mediated regulation of endothelial nitric oxide synthase and mitogen-activated protein kinase activities.

Somatostatin was reported to inhibit Kaposi's sarcoma (KS) cell (KS-Imm) xenografts through an antiangiogenic activity. Here, we show that somatostatin blocks growth of established KS-Imm tumors with the same efficacy as adriamycin, a clinically effective cytotoxic drug. Whereas KS-Imm cells do not express somatostatin receptors (SSTRs), endothelial cells express several SSTRs, in particular SSTR3. We investigated the molecular mechanisms and receptor specificity of somatostatin inhibition of angiogenesis. Somatostatin significantly inhibited angiogenesis in vivo in the matrigel sponge assay; this inhibition was mimicked by the SSTR3 agonist L-796778 and reversed by the SSTR3 antagonist BN81658, demonstrating involvement of SSTR3. In vitro experiments showed that somatostatin directly affected different endothelial cell line proliferation through a block of growth-factor-stimulated MAPK and endothelial nitric oxide (NO) synthase (eNOS) activities. BN81658 reversed somatostatin inhibition of cell proliferation, NO production, and MAPK activity, indicating that SSTR3 activation is required for the effects of somatostatin in vitro. Finally in vivo angiogenesis assays demonstrated that eNOS inhibition was a prerequisite for the antiangiogenic effects of somatostatin, because high concentrations of sodium nitroprusside, an NO donor, abolished the somatostatin effects. In conclusion, we demonstrate that somatostatin is a powerful antitumor agent in vivo that inhibits tumor angiogenesis through SSTR3-mediated inhibition of both eNOS and MAPK activities.

Somatostatin and its analog lanreotide inhibit the proliferation of dispersed human non-functioning pituitary adenoma cells in vitro.

OBJECTIVE: Somatostatin is a powerful inhibitor of hormone secretion and cell proliferation. Treatment with somatostatin analogs in humans causes a reduction in size and secretory activity of some endocrine tumors, including somatotropic pituitary adenomas. Less studied are the effects of somatostatin agonists on non-functioning pituitary adenomas (NFPAs). In this study we characterized the effects of somatostatin and its analog lanreotide on the proliferation of NFPAs in vitro and the intracellular mechanisms involved. DESIGN: Twenty-three NFPA post-surgical specimens were analyzed for somatostatin receptor (SSTR) expression and 12 of them were cultured in vitro to study somatostatin's effects on cell proliferation, assessed by means of [(3)H]thymidine uptake, and the intracellular signaling. RESULTS: One or more SSTR subtypes were expressed in 90% of the adenomas tested. Somatostatin and lanreotide treatment inhibited phorbol myristate acetate (PMA)-induced cell proliferation. Vanadate pretreatment reversed somatostatin and lanreotide inhibition of PMA-induced DNA synthesis suggesting an involvement of tyrosine phosphatase in this effect. In the only adenoma tested, somatostatin directly induced a tyrosine phosphatase activity. Somatostatin and lanreotide caused also a significant inhibition of voltage-sensitive calcium channel activity induced by 40mmol/l K(+) depolarization in microfluorimetric analysis. CONCLUSIONS: These data show that somatostatin and lanreotide inhibit human NFPA cell proliferation in vitro, and suggest that activation of tyrosine phosphatases and inhibition of the activity of voltage-dependent calcium channels may represent intracellular signals mediating this effect.

Expression in E. coli and purification of recombinant fragments of wild type and mutant human prion protein.

Prion diseases are fatal neurodegenerative disorders of the CNS of men and animals, characterized by spongiform degeneration of the CNS, astrogliosis and deposition of amyloid into the brain. The conversion of a cellular glycoprotein (the prion protein, PrP(C)) into an altered isoform (the prion scrapie, PrP(Sc)), which accumulates within the brain tissue by virtue of its resistance to the intracellular catabolism, is currently believed to represent the etiologic agent responsible for these diseases. Synthetic or recombinant polypeptides are commonly used to elucidate the mechanism of proteins involved in neurodegenerative diseases. Here we describe a procedure, which allows the synthesis and purification in its native folding, of the human prion protein fragment 90-231, corresponding to the protease resistant core of PrP(Sc). We synthesized the polypeptides 90-231 of both the wild type and the E200K mutant isoforms of PrP. Using a gluthatione S-transferase (GST) fusion protein approach, milligram amounts of polypeptides were obtained after expression in E. coli. The recovery of the purified fusion protein was monitored following the evaluation of the GST activity. The PrP fragment was released from the fusion protein immobilized on a glutathione-coupled agarose resin by direct cleavage with thrombin. The recombinant protein was identified by comassie stained acrylamide gel and by immunoblotting employing a monoclonal anti-PrP antibody. The peptide purified by gel filtration chromatography showed mainly an alpha-helix structure, as analysed by circular dichroism (CD) and an intact disulfide bridge. The same procedure was also successfully employed to synthesize and purify the E200K mutant PrP fragment.

Prion protein fragment 106-126 induces a p38 MAP kinase-dependent apoptosis in SH-SY5Y neuroblastoma cells independently from the amyloid fibril formation.

Prion diseases are neurodegenerative disorders of the central nervous system of humans and animals, characterized by spongiform degeneration of the central nervous system, astrogliosis, and deposition of amyloid into the brain. The conversion of a cellular glycoprotein (prion protein, PrP(C)) into an altered isoform (PrP(Sc)) has been proposed to represent the causative event responsible for these diseases. The peptide corresponding to the residues 106-126 of PrP sequence (PrP106-126) is largely used to explore the neurotoxic mechanisms underlying the prion diseases. We investigated the intracellular signaling responsible for PrP106-126-dependent cell death in the SH-SY5Y human neuroblastoma cell line. In these cells, PrP106-126 treatment induced apoptotic cell death and the activation of caspase-3. The p38 MAP-kinase blockers (SB203580 and PD169316) prevented the apoptotic cell death evoked by PrP106-126 and Western blot analysis revealed that the exposure of the cells to the peptide induced p38 activation. However, whether the neuronal toxicity of PrP106-126 is caused by a soluble or fibrillar form of this peptide is still unknown. In this study, we correlated the structural state of this peptide with its neurotoxicity. We show that the two conserved glycines in position 114 and 119 prevent the peptide to assume a structured conformation, favoring its aggregation in amyloid fibrils. The substitution of both glycines with alanine residues (PrP106-126AA) generates a soluble nonamyloidogenic peptide, that retained its toxic properties when incubated with neuroblastoma cells. These data show that the amyloid aggregation is not necessary for the induction of the toxic effects of PrP106-126.

Somatostatin receptor 1 (SSTR1)-mediated inhibition of cell proliferation correlates with the activation of the MAP kinase cascade: role of the phosphotyrosine phosphatase SHP-2.

The mitogen activated protein (MAP) kinase cascade represents one of the major regulator of cell growth by hormones and growth factors. However, although the activation of this intracellular pathway has been often regarded as mediator of cell proliferation, in many cell types the increase in MAP kinase (also called extra-cellular signal regulated kinase: ERK) activity may result in cell growth arrest, depending on the length or the intensity of the stimulation. In this review we examine recent data concerning the effects of somatostatin on the MAP kinase cascade through one of its major receptor subtype, the somatostatin receptor 1 (SSTR1), stably expressed in CHO-K1 cells. Somatostatin inhibits the proliferative effects of basic FGF (bFGF) in CHO-SSTR1 cell line. However, in these cells, somatostatin robustly activates the MAP kinase and augments bFGF-induced stimulation of ERK. We show that the activation of ERK via SSTR1 is mediated by the betagamma subunit of a pertussis toxin-sensitive G-protein and requires both the small G protein Ras and the serine/threonine kinase Raf-1. Moreover the phosphatidyl inositol-3kinase and the cytosolic tyrosine kinase c-src participate in the signal transduction regulated by SSTRI to activate ERK, as well as it is involved the protein tyrosine phosphatase (PTP) SHP-2. Previous studies have suggested that somatostatin-stimulated PTP activity mediates the growth inhibitory actions of somatostatin, in CHO-SSTR1 cells. Thus, the activation of SHP-2 by SSTR1 may mediate the antiproliferative activity of somatostatin. SHP-2 may. in turn, regulate the activity of kinases upstream of ERK that require tyrosine dephosphorylation to be activated, such as c-src. Finally, the synergism between somatostatin and bFGF in the activation of ERK results in an increased expression of the cyclin-dependent kinase inhibitor p21cip/WAF1 as molecular effector of the antiproliferative activity of somatostatin.

Intracellular mechanisms mediating the neuronal death and astrogliosis induced by the prion protein fragment 106-126.

Prion encephalopathies include fatal diseases of the central nervous system of men and animals characterized by nerve cell loss, glial proliferation and deposition of amyloid fibrils into the brain. During these diseases a cellular glycoprotein (the prion protein, PrP(C)) is converted, through a not yet completely clear mechanism, in an altered isoform (the prion scrapie, PrP(Sc)) that accumulates within the brain tissue by virtue of its resistance to the intracellular catabolism. PrP(Sc) is believed to be responsible for the neuronal loss that is observed in the prion disease. The PrP 106-126, a synthetic peptide that has been obtained from the amyloidogenic portion of the prion protein, represents a suitable model for studying the pathogenic role of the PrP(Sc), retaining, in vitro, some characteristics of the entire protein, such as the capability to aggregate in fibrils, and the neurotoxicity. In this work we present the results we have recently obtained regarding the action of the PrP 106-126 in different cellular models. We report that the PrP 106-126 induces proliferation of cortical astrocytes, as well as degeneration of primary cultures of cortical neurons or of neuroectodermal stable cell lines (GH(3) cells). In particular, these two opposite effects are mediated by the same attitude of the peptide to interact with the L-type calcium channels: in the astrocytes, the activity of these channels seems to be activated by PrP 106-126, while, in the cortical neurons and in the GH(3) cells, the same treatment causes a blockade of these channels causing a toxic effect.

Synthesis, characterization and in vitro evaluation of dimethyl-beta-cyclodextrin-4-biphenylylacetic acid conjugate.

Biphenylylacetic acid (BPAA) was linked to the free hydroxyl group of 2,6-di-O-methyl-beta-Cyclodextrin (DM-beta-CyD) through an ester linkage to obtain the site specific release of the drug to the colon. The conjugate at 1:1 mole ratio was separated from the reaction mixture by semipreparative reverse-phase HPLC and characterized by 1H-NMR, 13C-NMR, IR spectroscopy, mass spectrometry and elemental analysis. Chemico-physical characteristics, such as water solubility and dissolution rate, were evaluated comparatively to the BPAA-DM-beta-CyD inclusion complex. Hydrolysis rates were investigated in media simulating gastro-intestinal fluids and at pH 7.4 in the presence of porcine liver esterase. A rapid release of the drug was observed at acid pH value. In all cases a first order kinetic was observed, characterized by t1/2 value of 1.19, 19 and 4 h for chemical hydrolysis at pH 1.1, at pH 7.4 and enzymatic hydrolysis, respectively. In vitro permeation studies through caco-2 cells confirmed the ability of DM-beta-CyD to increase the absorption of included BPAA. A slow permeation was observed for the drug conjugate to DM-beta-CyD due to the slow release of BPAA.

Biomembrane model interaction and percutaneous absorption of papaverine through rat skin: effects of cyclodextrins as penetration enhancers.

The effects of different concentrations of beta-cyclodextrin (beta-CyD), hydroxypropyl-beta-cyclodextrin (HP-beta-CyD) and 2,6-di-O-methyl-beta-cyclodextrin (DM-beta-CyD) on percutaneous absorption of papaverine hydrochloride (PAP) were investigated. Abdominal rat skin mounted in Franz cells was used for in vitro experiments. To evaluate CyD interaction with a bilayer structure model, dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and DPPC-Chol (8:2 mole ratio) vesicles were used. CyD vesicle interaction was evaluated by differential scanning calorimetry. Permeation through rat skin and calorimetric experiments demonstrated that at low concentrations DM-beta-CyD shows higher enhancer activity as a possible result of a perturbing action on the skin by a complexation of its lipid components, but at higher concentrations HP-beta-CyD is the most effective. By considering that HP-beta-CyD presents a very moderate destabilizing action on the skin, we conclude that a 10% aqueous solution of this macrocycle appears to be the most suitable transdermal absorption enhancer for PAP.

[Somatostatinergic control of Kaposi's sarcoma growth through the inhibition of angiogenesis]. / La somatostatina inibisce la crescita di sarcoma di Kaposi in topi nudi mediante l'inibizione della neoangiogenesi.

Somatostatin and its analogs are active in the inhibition of the proliteration of sst receptor positive endocrine neoplasms, however their activity and mechanism in non-endocrine tumors is not clear. Somatostatin effectively inhibited the growth of a Kaposi's sarcoma xenograft in nude mice, yet in vitro the tumor cells did not express any known somatostatin receptors and were not growth inhibited by somatostatin. Histological examination revealed limited vascularization in the somatostatin treated tumors as compared to the controls. Somatostatin was a potent inhibitor of angiogenesis in an in vivo assay. In vitro, somatostatin inhibited endothelial cell growth and invasion. Migration of monocytes, important mediators of the angiogenic cascade, was also inhibited by somatostatin. Both cells types expressed somatostatin receptor mRNAs. These data demonstrate that somatostatin is a potent anti-tumor angiogenesis compound directly affecting both endothelial and monocytic cells. The debated function of somatostatin in tumor treatment and the design of therapeutic protocols should be reexamined considering these data.

[The activation of the phosphotyrosine phosphatase eta is responsible for the somatostatin inhibition of PCCl3 thyroid cell proliferation]. / L'attivazione della fosfotirosino fosfatasi eta è responsabile dell'inibizione della proliferazione delle cellule tiroidee PCC13 ad opera della somatostatin.

BACKGROUND: This study was aimed to identify possible intracellular effectors of the somatostatin (SST) antiproliferative activity, in PCCl3 thyroid cells. METHODS: To prove the involvement of r-PTPeta in SST's effect, we studied th proliferative activity of subclones of PCCl3 cells that do or do not express this PTP. RESULTS: SST inhibited PCCl3 TSH+insulin-dependent cell proliferation through the induction of a phosphotyrosine phosphatase (PTP) activity, detected using the synthetic substrate pNPP (+150%, p<0.01). Conversely, PCCl3 cells stably expressing the v-mos oncogene (PCmos) were completely insensitive to SST antiproliferative effects due to the incapability of SST to increase PTP activity, that correlated with the abolishment of the expression of the receptor-like PTP, r-PTPeta. In the cells in which r-PTPeta was transfected (PCmos/ PTPeta) SST inhibited cell proliferation showing a dose-dependence similar to that observed in PCCl3 cells. Conversely, the transfection of a catalytically inactive mutant of r-PTPeta did not restore the responsivity to SST. Also in PCmos/PTPeta cells SST, treatment increased membrane PTP activity. CONCLUSIONS: SST inhibition of PCC13 cell proliferation requires the activation of r-PTPeta.

Diastereo- and enantioselective synthesis of 1'-C-branched N,O-nucleosides.

A synthetic approach towards 1'-C-branched N,O-nucleosides is reported, based on 1,3-dipolar cycloaddition of ethoxycarbonylnitrone. The asymmetric version of the process exploits the presence of a chiral auxiliary at the carbon atom of nitrone and leads to beta-D and beta-L nucleosides in good yields.

[Correlative evaluation of electrocardiographic changes and spirometric parameters in pulmonary cardiopathy secondary to chronic obstructive bronchopneumopathy]. / Valutazione correlativa tra modifiche elettrocardiografiche e parametri spirometrici nella cardiopatia polmonare secondaria a broncopneumopatie croniche ostruttive.

EKG is not considered a sensitive procedure for diagnosis of chronic pulmonary heart disease (CPHD). We performed a correlative study between EKG signs and spirometry in chronic obstructive pulmonary disease (COPD). Fifty-six consecutive in-patients (M 52, F 4, age 59.1 +/- 13.1 yrs) with COPD and echocardiographic/roentgenologic signs of right ventricular enlargement were studied. An EKG score system (0-6) was developed considering: 1) AQRS greater than or equal to 90 degrees; 2) P waves greater than or equal to 2.5 mm; 3) R wave V1 greater than or equal to 7 mm or R/S V1 greater than or equal to 1; 4) R/S V5 less than or equal to 1; 5) RV strain pattern; 6) RBBB. Significant (p less than 0.01) linear correlations were observed between: a) AQRS vs FEV1 (r = -0.59); b) AQRS vs FEV1/FVC% (r = -0.61); c) R/S V5 vs FEV1, (r = +0.38); d) EKG score vs FEV1 (r = -0.42); e) EKG score vs FEV1/FVC% (r = -0.40). Significant relationship between EKG score and spirometry suggests that this criterium can be useful for a more reliable ecg diagnosis of CPHD.

Combined microwave thermal ablation and liver resection for single step treatment of otherwise unresectable colorectal liver metastases; a monoistitutional experiences.

OBJECTIVE: Over half of colorectal cancer patients will develop liver metastases. BACKGROUND: Thermal ablation with or without associated liver resection for colorectal hepatic metastasis has been suggested as an alternative method to improve survival if radical surgical resection is not achievable. A retrospective case series of patients treated with microwave ablation(MWA) associated with hepatic resection in one step procedure, was reviewed to analyze the clinical outcome. MATERIALS AND METHODS: In a group of 40 patients surgically cured for liver tumors in our Department, 5 patients with technically unresectable disease underwent combined treatment LR-MWA. RESULTS: Four patients were treated with multiple segmentectomies and MWA and one patient received a left lobectomy (S2-S3) and MWA. Only 1 patient (20%) developed post surgical complication which was a liver abscess (grade II of Dindo classification). CONCLUSIONS: Hepatic resection combined with MWA expanded indications for operative treatment of multiple bilobar liver metastasis. This procedure promise to have good long-term outcomes.

Human PrP90-231-induced cell death is associated with intracellular accumulation of insoluble and protease-resistant macroaggregates and lysosomal dysfunction.

To define the mechanisms by which hPrP90-231 induces cell death, we analyzed its interaction with living cells and monitored its intracellular fate. Treatment of SH-SY5Y cells with fluorescein-5-isothiocyanate (FITC)-conjugated hPrP90-231 caused the accumulation of cytosolic aggregates of the prion protein fragment that increased in number and size in a time-dependent manner. The formation of large intracellular hPrP90-231 aggregates correlated with the activation of apoptosis. hPrP90-231 aggregates occurred within lysotracker-positive vesicles and induced the formation of activated cathepsin D (CD), indicating that hPrP90-231 is partitioned into the endosomal-lysosomal system structures, activating the proteolytic machinery. Remarkably, the inhibition of CD activity significantly reduced hPrP-90-231-dependent apoptosis. Internalized hPrP90-231 forms detergent-insoluble and SDS-stable aggregates, displaying partial resistance to proteolysis. By confocal microscopy analysis of lucifer yellow (LY) intracellular partition, we show that hPrP90-231 accumulation induces lysosome destabilization and loss of lysosomal membrane impermeability. In fact, although control cells evidenced a vesicular pattern of LY fluorescence (index of healthy lysosomes), hPrP90-231-treated cells showed diffuse cytosolic fluorescence, indicating LY diffusion through damaged lysosomes. In conclusion, these data indicate that exogenously added hPrP90-231 forms intralysosomal deposits having features of insoluble, protease-resistant aggregates and could trigger a lysosome-mediated apoptosis by inducing lysosome membrane permeabilization, followed by the release of hydrolytic enzymes.