Accumulation of Mutated Maize Zeins in Transgenic Forage Legumes

Accumulation of zeins, the endosperm storage proteins of maize, in a heterologous plant expression system was attempted. Plants of birdsfoot trefoil (Lotus corniculatus) and alfalfa (Medicago sativa) were transformed by Agrobacterium with binary vectors harboring genes that code for γ-zein and β-zein, two proteins rich in sulphur amino acids. Adding the ER retention signal KDEL to the C-terminal domain modified zein polypeptides. Our long-term goal was to improve birdsfoot trefoil and alfalfa forage quality. Significant levels of γ- zein:KDEL and β-zein:KDEL were detected in primary transformants of birdsfoot trefoil. Moreover, alfalfa plants expressing γ-zein:KDEL in the leaves were obtained. γ-zein:KDEL accumulated in spherical or elliptical electron-dense bodies of birdsfoot trefoil leaves. The protein bodies were present in the cytoplasm of either mesophyll cells or epidermis cells

Sull'approccio umano-centrico all'intelligenza artificiale. Riflessioni a margine del "Progetto europeo di orientamenti etici per una IA affidabile"

Il giurista \ue8 sempre chiamato a compiere delle scelte: in ci\uf2 consiste la sua attivit\ue0, nel valutare per decidere . Tuttavia, vi sono dei momenti storicamente determinati ove que- ste scelte si rivelano risolutive per la conservazione e l\u2019evoluzione della civilt\ue0. In questi momenti, che segnano indelebilmente la storia dell\u2019uomo, il giurista ha la responsabilit\ue0 di compiere valutazioni concrete e assumere decisioni ragionevoli, secondo la filosofia che \ue8 nel diritto : non una qualsiasi etica n\ue9 linee guida, ma valori normativi di civilt\ue0 giuridica

L'impatto delle nuove tecnologie sul diritto

Il rapporto tra interpretazione giuridica e nuove tecnologie richiama nuovamente l\u2019attenzione sulla valutazione del \u2018tempo\u2019,sul cambiamento dei modi di esplicarsi dell\u2019attivit\ue0 umana e nella stessa visione della realt\ue0,sollecitando il giurista contemporaneo a sottoporre a revisione le basi del proprio mestier

H3 histamine receptor-mediated activation of protein kinase calpha inhibits the growth of cholangiocarcinoma in vitro and in vivo

Histamine regulates functions via four receptors (HRH1, HRH2, HRH3, and HRH4). The D-myo-inositol 1,4,5-trisphosphate (IP(3))/Ca(2+)/protein kinase C (PKC)/mitogen-activated protein kinase pathway regulates cholangiocarcinoma growth. We evaluated the role of HRH3 in the regulation of cholangiocarcinoma growth. Expression of HRH3 in intrahepatic and extrahepatic cell lines, normal cholangiocytes, and human tissue arrays was measured. In Mz-ChA-1 cells stimulated with (R)-(alpha)-(-)-methylhistamine dihydrobromide (RAMH), we measured (a) cell growth, (b) IP(3) and cyclic AMP levels, and (c) phosphorylation of PKC and mitogen-activated protein kinase isoforms. Localization of PKC alpha was visualized by immunofluorescence in cell smears and immunoblotting for PKC alpha in cytosol and membrane fractions. Following knockdown of PKC alpha, Mz-ChA-1 cells were stimulated with RAMH before evaluating cell growth and extracellular signal-regulated kinase (ERK)-1/2 phosphorylation. In vivo experiments were done in BALB/c nude mice. Mice were treated with saline or RAMH for 44 days and tumor volume was measured. Tumors were excised and evaluated for proliferation, apoptosis, and expression of PKC alpha, vascular endothelial growth factor (VEGF)-A, VEGF-C, VEGF receptor 2, and VEGF receptor 3. HRH3 expression was found in all cells. RAMH inhibited the growth of cholangiocarcinoma cells. RAMH increased IP(3) levels and PKC alpha phosphorylation and decreased ERK1/2 phosphorylation. RAMH induced a shift in the localization of PKC alpha expression from the cytosolic domain into the membrane region of Mz-ChA-1 cells. Silencing of PKC alpha prevented RAMH inhibition of Mz-ChA-1 cell growth and ablated RAMH effects on ERK1/2 phosphorylation. In vivo, RAMH decreased tumor growth and expression of VEGF and its receptors; PKC alpha expression was increased. RAMH inhibits cholangiocarcinoma growth by PKC alpha-dependent ERK1/2 dephosphorylation. Modulation of PKC alpha by histamine receptors may be important in regulating cholangiocarcinoma growth. (Mol Cancer Res 2009;7(10):1704-13

Chronic Cerebrospinal Venous Insufficiency (CCSVI) IN Meniere Disease. Case or Cause?

Abstract CCSVI is the acronym for Chronic Cerebrospinal Venous Insufficiency, initially described by P.Zamboni, as being strongly associated with multiple sclerosis (MS). It is a syndrome characterized by stenosis of the internal jugular veins (IJVs) and/or azygous vein (AZ) with opening of collaterals and insufficient drainage. Bavera PM carried out 823 Duplex exams on a control group of 60 patients without MS. As expected CCSVI was found only in few subjects of the control group, three, two females and one male, but all affected with Sudden Sensorineural Hearing Loss (SSHL). Successively, we reported a case of bilateral SSHL with vertigo, showing evidence of the CCSVI pattern at Duplex examination (not associated with MS). To the best of the authors' knowledge, this kind of association has never been reported. We studied 52 patients affected with cochleo-vestibular disturbances subdivided into two groups of out-patients:Definite unilateral Meniere (Men): 12 subjects (8 males and 4.females, mean age 41,6.yy) according to international AOO-HNS 1995 diagnostic criteria -No-Meniere (No-Men): 14 subjects (6.males and 8 females, mean age 44,7.yy) affected with unilateral cocleo-vestibular impairment A third group of subjects have been considered, as a "normal" group, 13 patients (8 females and 5 males, mean age 45,5 yy) affected with Benign Paroxismal Positioning Vertigo (BPPV) with cochlear involvement Asymmetrical artherious flow in VA or CA was revealed in 2 Men 9 no-Men and 1 BPPV, respectively 12,5 -60,7 -and 8,6 %. Differences between Men and NoMen and between each of this group with respect to BPPV were highly significant (p<0.001). Asymmetrical venous flow in IJV or VV was detected in 9 patients in MEN group and in 4 in no-MEN and 2 BPPV, respectively 79 -28,5 and 13 %. Differences between Men and No-Men and between each of this group with respect to BBV were highly significant (p<0.001

Histamine stimulates the proliferation of small and large cholangiocytes by activation of both IP3/Ca2+ and cAMP-dependent signaling mechanisms

Although large cholangiocytes exert their functions by activation of cyclic adenosine 3',5'-monophosphate (cAMP), Ca(2+)-dependent signaling regulates the function of small cholangiocytes. Histamine interacts with four receptors, H1-H4HRs. H1HR acts by Gαq activating IP(3)/Ca(2+), whereas H2HR activates Gα(s) stimulating cAMP. We hypothesize that histamine increases biliary growth by activating H1HR on small and H2HR on large cholangiocytes. The expression of H1-H4HRs was evaluated in liver sections, isolated and cultured (normal rat intrahepatic cholangiocyte culture (NRIC)) cholangiocytes. In vivo, normal rats were treated with histamine or H1-H4HR agonists for 1 week. We evaluated: (1) intrahepatic bile duct mass (IBDM); (2) the effects of histamine, H1HR or H2HR agonists on NRIC proliferation, IP(3) and cAMP levels and PKCα and protein kinase A (PKA) phosphorylation; and (3) PKCα silencing on H1HR-stimulated NRIC proliferation. Small and large cholangiocytes express H1-H4HRs. Histamine and the H1HR agonist increased small IBDM, whereas histamine and the H2HR agonist increased large IBDM. H1HR agonists stimulated IP(3) levels, as well as PKCα phosphorylation and NRIC proliferation, whereas H2HR agonists increased cAMP levels, as well as PKA phosphorylation and NRIC proliferation. The H1HR agonist did not increase proliferation in PKCα siRNA-transfected NRICs. The activation of differential signaling mechanisms targeting small and large cholangiocytes is important for repopulation of the biliary epithelium during pathologies affecting different-sized bile ducts

Inhibition of mapk signalling promotes cell cycle arrest and sensitises intrahepatic cholangiocarcinoma cells to chemotherapy

Introduction: Intrahepatic cholangiocarcinoma (ICC) is the second most common primary hepatic malignancy, accounting for approximately 15% of cases of primary liver cancer. Although new treatments have increased survival for many other cancers, including the more common primary hepatocellular carcinoma, treatment strategies and survival for patients with ICC have seen little improvement. Our previous studies suggest that the mitogen-activated protein kinase (MAPK) signalling plays a central role in the regulation of cell proliferation in human ICC. However the molecular mechanisms are poorly understood. In this study, we aim to explore whether inhibition of the MAPK pathway and its downstream effectors enhances the sensitisation of ICC cells to the chemotherapeutic agent cisplatinum. Method: We used a combinatorial approach of immunohistochemical and gene expression analyses to investigate the expression of MAPK-related genes in ICC tumours. Furthermore, by using in-vitroand in-vivoanalyses we have characterised the function of a novel MAPK downstream effector in ICC cells. Results: The expression of MAPK signalling was determined by immunohistochemical staining in tumour samples from a cohort of 14 ICC patients. High expression of phospho-activated MAPK was observed in 71.4% (10/14) of ICC cases as compared with surrounding nontumour tissue. Likewise, expression of JDP, a downstream effector of the MAPK signalling, was scored as high intensity in 64.3% (9/14). Strikingly, elevated expression of JDP transcripts was also observed in two independent cohorts of human ICC (n = 149 and n = 109 per group, respectively) compared to surrounding normal liver tissue. Consistent with the in-vivo analyses of human samples, immunoblotting analyses showed constitutive activation of MAPK and expression of JDP in ICC-derived cells (i.e. SG231, CCLP-1 and HuCCT1). Using loss-of-function analyses, we demonstrates that knockdown of JDP in ICC-derived cells resulted in cell cycle arrest and reduced expression of cell cycle regulators (i.e. cyclins), and had minimal effect on apoptosis. Chemical inhibition of JDP significantly sensitises ICC-derived cells to cisplatinum (P < 0.001). Conclusion: These results demonstrate that enhanced activation of MAPK signalling is important for ICC cell proliferation and suggest that targeting its downstream effectors is a potential therapeutic strategy for ICC