Prolactin stimulates the proliferation of normal female cholangiocytes by differential regulation of Ca2+-dependent PKC isoforms

Background

Prolactin promotes proliferation of several cells. Prolactin receptor exists as two isoforms: long and short, which activate different transduction pathways including the Ca²⁺-dependent PKC-signaling. No information exists on the role of prolactin in the regulation of the growth of female cholangiocytes. The rationale for using cholangiocytes from female rats is based on the fact that women are preferentially affected by specific cholangiopathies including primary biliary cirrhosis. We propose to evaluate the role and mechanisms of action by which prolactin regulates the growth of female cholangiocytes.

Results

Normal cholangiocytes express both isoforms (long and short) of prolactin receptors, whose expression increased following BDL. The administration of prolactin to normal female rats increased cholangiocyte proliferation. In purified normal female cholangiocytes, prolactin stimulated cholangiocyte proliferation, which was associated with increased [Ca²⁺]_i levels and PKCβ-I phosphorylation but decreased PKCα phosphorylation. Administration of an anti-prolactin antibody to BDL female rats decreased cholangiocyte proliferation. Normal female cholangiocytes express and secrete prolactin, which was increased in BDL rats. The data show that prolactin stimulates normal cholangiocyte growth by an autocrine mechanism involving phosphorylation of PKCβ-I and dephosphorylation of PKCα.

Conclusion

We suggest that in female rats: (i) prolactin has a trophic effect on the growth of normal cholangiocytes by phosphorylation of PKCβ-I and dephosphorylation of PKCα; and (iii) cholangiocytes express and secrete prolactin, which by an autocrine mechanism participate in regulation of cholangiocyte proliferation. Prolactin may be an important therapeutic approach for the management of cholangiopathies affecting female patients.     

Reduced biliverdin reductase-A levels are associated with early alterations of insulin signaling in obesity

Biliverdin reductase-A (BVR-A) is a serine/threonine/tyrosine kinase involved in the regulation of insulin signaling. In vitro studies have demonstrated that BVR-A is a substrate of the insulin receptor and regulates IRS1 by avoiding its aberrant activation, and in animal model of obesity the loss of hepatic BVR-A has been associated with glucose/insulin alterations and fatty liver disease. However, no studies exist in humans. Here, we evaluated BVR-A expression levels and activation in peripheral blood mononuclear cells (PBMC) from obese subjects and matched lean controls and we investigated the related molecular alterations of the insulin along with clinical correlates. We showed that BVR-A levels are significantly reduced in obese subjects and associated with a hyper-activation of the IR/IRS1/Akt/GSK-3β/AS160/GLUT4 pathway. Low BVR-A levels also associate with the presence of obesity, metabolic syndrome, NASH and visceral adipose tissue inflammation. These data suggest that the reduction of BVR-A may be responsible for early alterations of the insulin signaling pathway in obesity and in this context may represent a novel molecular target to be investigated for the comprehension of the process of insulin resistance development in obesity.     

AhR ligand aminoflavone suppresses α6-integrin–Src–Akt signaling to attenuate tamoxifen resistance in breast cancer cells

More than 40% of patients with luminal breast cancer treated with endocrine therapy agent tamoxifen demonstrate resistance. Emerging evidence suggests tumor initiating cells (TICs) and aberrant activation of Src and Akt signaling drive tamoxifen resistance and relapse. We previously demonstrated that aryl hydrocarbon receptor ligand aminoflavone (AF) inhibits the expression of TIC gene α6-integrin and disrupts mammospheres derived from tamoxifen-sensitive breast cancer cells. In the current study, we hypothesize that tamoxifen-resistant (TamR) cells exhibit higher levels of α6-integrin than tamoxifen-sensitive cells and that AF inhibits the growth of TamR cells by suppressing α6-integrin–Src–Akt signaling. In support of our hypothesis, TamR cells and associated mammospheres were found to exhibit elevated α6-integrin expression compared with their tamoxifen-sensitive counterparts. Furthermore, tumor sections from patients who relapsed on tamoxifen showed enhanced α6-integrin expression. Gene expression profiling from the TCGA database further revealed that basal-like breast cancer samples, known to be largely unresponsive to tamoxifen, demonstrated higher α6-integrin levels than luminal breast cancer samples. Importantly, AF reduced TamR cell viability and disrupted TamR mammospheres while concomitantly reducing α6-integrin messenger RNA and protein levels. In addition, AF and small interfering RNA against α6-integrin blocked tamoxifen-stimulated proliferation of TamR MCF-7 cells and further sensitized these cells to tamoxifen. Moreover, AF reduced Src and Akt signaling activation in TamR MCF-7 cells. Our findings suggest elevated α6-integrin expression is associated with tamoxifen resistance and AF suppresses α6-integrin–Src–Akt signaling activation to confer activity against TamR breast cancer.     

Evaluation of the FocalPoint GS system performance in an Italian population-based screening of cervical abnormalities

OBJECTIVE: To evaluate the FocalPoint Location-Guided Screening (FPGS) performance in computer-assisted primary screening of Papanicolaou-stained cervicovaginal smears. STUDY DESIGN: A total of 37,306 routine consecutive conventional Pap slides were prospectively processed on the FPGS. Each slid designated by the instrument as Review was reported according to results obtained using a GS Review Station. Subsequently, all slides under went conventional manual rapid screening and reported results were compared. RESULTS: Of the slides initially submitted to the FPGS, 34,004 (91.15%) were qualified for scanning. Within these slides, the system classified 7,399 (21.8%) as needing No Further Review and ranked to Review the remaining 26,605 (78.2%). Of the 418 cellular abnormalities found, 409 were classified for Review by FPGS and 9 minor grade lesions were classified in the "No Further Review" population. Overall, 352 (86%) of atypical squamous cell (ASC)+ were ranked in high-score quintiles, including 96 (94%) of the 102 high-grade squamous intraepithelial lesion (HSIL) or worse. Location-guided software identified cellular abnormalities, in the automatically selected fields of view, in 378 (92%) of the ranked abnormal slides, showing a sensitivity > 95% on SILs. CONCLUSION: Slide ranking and location-guided screening features are of value in detecting and triaging abnormal smears.     

Are novel ecosystems suitable habitats for native birds? Birds’ association with spontaneous vegetation patches in the argentinean pampas

Biodiversity and Conservation - Novel ecosystems are characterized by species compositions and relative abundances that differ from those that occurred previously and have the potential for...     

The Toluene o-Xylene Monooxygenase Enzymatic Activity for the Biosynthesis of Aromatic Antioxidants

Monocyclic phenols and catechols are important antioxidant compounds for the food and pharmaceutic industries; their production through biotransformation of low-added value starting compounds is of major biotechnological interest. The toluene o-xylene monooxygenase (ToMO) from Pseudomonas sp. OX1 is a bacterial multicomponent monooxygenase (BMM) that is able to hydroxylate a wide array of aromatic compounds and has already proven to be a versatile biochemical tool to produce mono- and dihydroxylated derivatives of aromatic compounds. The molecular determinants of its regioselectivity and substrate specificity have been thoroughly investigated, and a computational strategy has been developed which allows designing mutants able to hydroxylate non-natural substrates of this enzyme to obtain high-added value compounds of commercial interest. In this work, we have investigated the use of recombinant ToMO, expressed in cells of Escherichia coli strain JM109, for the biotransformation of non-natural substrates of this enzyme such as 2-phenoxyethanol, phthalan and 2-indanol to produce six hydroxylated derivatives. The hydroxylated products obtained were identified, isolated and their antioxidant potential was assessed both in vitro, using the DPPH assay, and on the rat cardiomyoblast cell line H9c2. Incubation of H9c2 cells with the hydroxylated compounds obtained from ToMO-catalyzed biotransformation induced a differential protective effect towards a mild oxidative stress induced by the presence of sodium arsenite. The results obtained confirm once again the versatility of the ToMO system for oxyfunctionalization reactions of biotechnological importance. Moreover, the hydroxylated derivatives obtained possess an interesting antioxidant potential that encourages the use of the enzyme for further functionalization reactions and their possible use as scaffolds to design novel bioactive molecules.     

Sensitivity of Human Intrahepatic Cholangiocarcinoma Subtypes to Chemotherapeutics and Molecular Targeted Agents: A Study on Primary Cell Cultures

We investigated the sensitivity of intrahepatic cholangiocarcinoma (IHCCA) subtypes to chemotherapeutics and molecular targeted agents. Primary cultures of mucin- and mixed-IHCCA were prepared from surgical specimens (N. 18 IHCCA patients) and evaluated for cell proliferation (MTS assay) and apoptosis (Caspase 3) after incubation (72 hours) with increasing concentrations of different drugs. In vivo, subcutaneous human tumor xenografts were evaluated. Primary cultures of mucin- and mixed-IHCCA were characterized by a different pattern of expression of cancer stem cell markers, and by a different drug sensitivity. Gemcitabine and the Gemcitabine-Cisplatin combination were more active in inhibiting cell proliferation in mixed-IHCCA while Cisplatin or Abraxane were more effective against mucin-IHCCA, where Abraxane also enhances apoptosis. 5-Fluoracil showed a slight inhibitory effect on cell proliferation that was more significant in mixed- than mucin-IHCCA primary cultures and, induced apoptosis only in mucin-IHCCA. Among Hg inhibitors, LY2940680 and Vismodegib showed slight effects on proliferation of both IHCCA subtypes. The tyrosine kinase inhibitors, Imatinib Mesylate and Sorafenib showed significant inhibitory effects on proliferation of both mucin- and mixed-IHCCA. The MEK 1/2 inhibitor, Selumetinib, inhibited proliferation of only mucin-IHCCA while the aminopeptidase-N inhibitor, Bestatin was more active against mixed-IHCCA. The c-erbB2 blocking antibody was more active against mixed-IHCCA while, the Wnt inhibitor, LGK974, similarly inhibited proliferation of mucin- and mixed-IHCCA. Either mucin- or mixed-IHCCA showed high sensitivity to nanomolar concentrations of the dual PI3-kinase/mTOR inhibitor, NVP-BEZ235. In vivo, in subcutaneous xenografts, either NVP-BEZ235 or Abraxane, blocked tumor growth. In conclusion, mucin- and mixed-IHCCA are characterized by a different drug sensitivity. Cisplatin, Abraxane and the MEK 1/2 inhibitor, Selumetinib were more active against mucin-IHCCA while, Gemcitabine, Gemcitabine-Cisplatin combination, the c-erbB2 blocking antibody and bestatin worked better against mixed-IHCCA. Remarkably, we identified a dual PI3-kinase/mTOR inhibitor that both in vitro and in vivo, exerts dramatic antiproliferative effects against both mucin- and mixed-IHCCA.     

Antimicrobial activity of Myrtus communis L. water-ethanol extract against meat spoilage strains of Brochothrix thermosphacta and Pseudomonas fragi in vitro and in meat

The antimicrobial activity of hydro-alcoholic extract of Myrtus communis L. was investigate in vitro and in situ against different meat spoilage biotypes of Pseudomonas fragi and Brochothrix thermosphacta. Water-ethanol myrtle extract (WEME) was prepared from myrtle leaves and used to study the antimicrobial activity, Minimum Inhibitory Concentration (MIC) and Minimum Lethal Concentration (MLC). Naturally contaminated ground beef was used to evaluate in situ antibacterial activity of freeze-dried myrtle extract at different concentrations. In vitro antimicrobial activity of WEME was significantly more effective against B. thermosphacta than P. fragi strains. MIC and MLC values were in the range of 12.5–50 and 25–100 mg DM/ml, respectively, for B. thermosphacta and P. fragi strains. In situ results showed that the microbial population, except for Pseudomonas spp., decreased significantly in ground meat with added 5 % of freeze-dried myrtle extract. In conclusion, the findings demonstrate the effectiveness of myrtle extract to control or prevent the proliferation of meat spoilage bacteria.