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Androgens stimulate neuronal differentiation of PC12 cells by nontranscriptional action of the endogenous androgen receptor (AR). AR is also required for NGF-induced neuritogenesis of PC12. Androgens or NGF trigger AR association with filamin and TrkA, TrkA interaction with PI3-K δ, and activation of PI3-K δ and Rac., Steroids and growth factors control neuronal development through their receptors under physiological and pathological conditions. We show that PC12 cells harbor endogenous androgen receptor (AR), whose inhibition or silencing strongly interferes with neuritogenesis stimulated by the nonaromatizable synthetic androgen R1881 or NGF. This implies a role for AR not only in androgen signaling, but also in NGF signaling. In turn, a pharmacological TrkA inhibitor interferes with NGF- or androgen-induced neuritogenesis. In addition, androgen or NGF triggers AR association with TrkA, TrkA interaction with PI3-K δ, and downstream activation of PI3-K δ and Rac in PC12 cells. Once associated with AR, filamin A (FlnA) contributes to androgen or NGF neuritogenesis, likely through its interaction with signaling effectors, such as Rac. This study thus identifies a previously unrecognized reciprocal cross-talk between AR and TrkA, which is controlled by β1 integrin. The contribution of FlnA/AR complex and PI3-K δ to neuronal differentiation by androgens and NGF is also novel. This is the first description of AR function in PC12 cells.

Osteosarcoma (OS) is a primary highly malignant tumor of bone, affecting predominately adolescents and young adults between 10 and 20 years of age. OS is characterized by an extremely aggressive clinical course, with a rapid development of metastasis to the lung and distant bones.

Contains fulltext : 168891.pdf (Publisher’s version ) (Open Access) Deregulation of epigenetic mechanisms, including microRNA, contributes to leukemogenesis and drug resistance by interfering with cancer-specific molecular pathways. Here, we show that the balance between miR-194-5p and its newly discovered target BCL2-associated transcription factor 1 (BCLAF1) regulates differentiation and survival of normal hematopoietic progenitors. In acute myeloid leukemias this balance is perturbed, locking cells into an immature, potentially /`immortal/' state. Enhanced expression of miR-194-5p by treatment with the histone deacetylase inhibitor SAHA or by exogenous miR-194-5p expression re-sensitizes cells to differentiation and apoptosis by inducing BCLAF1 to shuttle between nucleus and cytosol. miR-194-5p/BCLAF1 balance was found commonly deregulated in 60 primary acute myeloid leukemia patients and was largely restored by ex vivo SAHA treatment. Our findings link treatment responsiveness to re-instatement of miR-194-5p/BCLAF1 balance. 11 p.

Objective. Alkaptonuria (AKU) is an ultra-rare autosomal recessive disease that currently lacks an appropriate therapy. Recently we provided experimental evidence that AKU is a secondary serum amyloid A (SAA)-based amyloidosis. The aim of the present work was to evaluate the use of antioxidants to inhibit SAA amyloid and pro-inflammatory cytokine release in AKU. Methods. We adopted a human chondrocytic cell AKU model to evaluate the anti-amyloid capacity of a set of antioxidants that had previously been shown to counteract ochronosis in a serum AKU model. Amyloid presence was evaluated by Congo red staining. Homogentisic acid-induced SAA production and pro-inflammatory cytokine release (overexpressed in AKU patients) were evaluated by ELISA and multiplex systems, respectively. Lipid peroxidation was evaluated by means of a fluorescence-based assay. Results. Our AKU model allowed us to prove the efficacy of ascorbic acid combined with N-acetylcysteine, taurine, phytic acid and lipoic acid in significantly inhibiting SAA production, pro-inflammatory cytokine release and membrane lipid peroxidation. Conclusion. All the tested antioxidant compounds were able to reduce the production of amyloid and may be the basis for establishing new therapies for AKU amyloidosis.

Alkaptonuria (AKU) is an ultra-rare disease developed from the lack of homogentisic acid oxidase activity, causing homogentisic acid (HGA) accumulation that produces a HGA-melanin ochronotic pigment, of unknown composition. There is no therapy for AKU. Our aim was to verify if AKU implied a secondary amyloidosis. Congo Red, Thioflavin-T staining and TEM were performed to assess amyloid presence in AKU specimens (cartilage, synovia, periumbelical fat, salivary gland) and in HGA-treated human chondrocytes and cartilage. SAA and SAP deposition was examined using immunofluorescence and their levels were evaluated in the patients' plasma by ELISA. 2D electrophoresis was undertaken in AKU cells to evaluate the levels of proteins involved in amyloidogenesis. AKU osteoarticular tissues contained SAA-amyloid in 7/7 patients. Ochronotic pigment and amyloid co-localized in AKU osteoarticular tissues. SAA and SAP composition of the deposits assessed secondary type of amyloidosis. High levels of SAA and SAP were found in AKU patients' plasma. Systemic amyloidosis was assessed by Congo Red staining of patients' abdominal fat and salivary gland. AKU is the second pathology after Parkinson's disease where amyloid is associated with a form of melanin. Aberrant expression of proteins involved in amyloidogenesis has been found in AKU cells. Our findings on alkaptonuria as a novel type II AA amyloidosis open new important perspectives for its therapy, since methotrexate treatment proved to significantly reduce in vitro HGA-induced A-amyloid aggregates., Highlights ► Alkaptonuria (no therapy) is a novel type II secondary amyloidosis. ► Ochronotic melanin-based pigment and SAA/SAP amyloid co-localize in Alkaptonuria. ► High levels of SAA and SAP were found in AKU patients' plasma. ► Systemic amyloidosis was assessed in patients' abdominal fat and salivary gland. ► Methotrexate reduces (- 97%) HGA-induced A-amyloid aggregates.

Alkaptonuria (AKU) results from defective homogentisate1,2-dioxygenase (HGD), causing degenerative arthropathy. The deposition of ochronotic pigment in joints is so far attributed to homogentisic acid produced by the liver, circulating in the blood and accumulating locally. Human normal and AKU osteoarticular cells were tested for HGD gene expression by RT-PCR, mono- and 2D-Western blotting. HGD gene expression was revealed in chondrocytes, synoviocytes, osteoblasts. Furthermore, HGD expression was confirmed by Western blotting, that also revealed the presence of five enzymatic molecular species. Our findings indicate that AKU osteoarticular cells produce the ochronotic pigment in loco and this may strongly contribute to induction of ochronotic arthropathy. J. Cell. Physiol. 227: 3254–3257, 2012. © 2011 Wiley Periodicals, Inc.

Correction to: Leukemia (2017) 31, 2315–2325; doi:10.1038/leu.2017.64; published online 10 March 2017 Following the publication of this article, the authors noted a mistake in the author affiliations. 4. Centre for Research in Molecular Medicine, The University of Lahore, Lahore, Pakistan Should be replaced with

Osteosarcoma (OS) is a primary highly malignant tumor of bone, affecting predominately adolescents and young adults between 10 and 20 years of age. OS is characterized by an extremely aggressive clinical course, with a rapid development of metastasis to the lung and distant bones.

Altered expression and activity of histone deacetylases (HDACs) have been correlated with tumorigenesis. Inhibitors of HDACs (HDACi) induce acetylation of histone and non-histone proteins affecting gene expression, cell cycle progression, cell migration, terminal differentiation and cell death. Here, we analyzed the regulation of ARHGEF3, a RhoA-specific guanine nucleotide exchange factor, by the HDACi MS275 (entinostat). MS275 is a well-known benzamide-based HDACi, which induces differentiation of the monoblastic-like human histiocytic lymphoma cell line U937 to monocytes/macrophages. Incubation of U937 cells with MS275 resulted in an up regulation of ARHGEF3, followed by a significant enhancement of the marker of macrophage differentiation CD68. ARHGEF3 protein is primarily nuclear, but MS275 treatment rapidly induced its translocation into the cytoplasm. ARHGEF3 cytoplasmic localization is associated with activation of the RhoA/Rho-associated Kinase (ROCK) pathway. In addition to cytoskeletal rearrangements orchestrated by RhoA, we showed that ARHGEF3/RhoA-dependent signals involve activation of SAPK/JNK and then Elk1 transcription factor. Importantly, MS275-induced CD68 expression was blocked by exposure of U937 cells to exoenzyme C3 transferase and Y27632, inhibitors of Rho and ROCK respectively. Moreover, ARHGEF3 silencing prevented RhoA activation leading to a reduction in SAPK/JNK phosphorylation, Elk1 activation and CD68 expression, suggesting a crucial role for ARHGEF3 in myeloid differentiation. Taken together, our results demonstrate that ARHGEF3 modulates acute myeloid leukemia differentiation through activation of RhoA and pathways directly controlled by small GTPase family proteins. The finding that GEF protein modulation by HDAC inhibition impacts on cell differentiation may be important for understanding the antitumor mechanism(s) by which HDACi treatment stimulates differentiation in cancer.

Alkaptonuria (AKU) is an ultra-rare disease developed from the lack of homogentisate 1,2-dioxygenase activity, causing an accumulation of homogentisic acid (HGA) in pigmented deposits (ochronosis) in connective tissues, especially in the joints and heart. AKU is mostly asymptomatic in early life, with arthropathy and cardiovascular symptoms appearing in later decades of life. Cardiovascular involvement has been described in as many as 40% of AKU patients (mean and median age of detection being 54 and 52 years, respectively) [1]. Alkaptonuric ochronosis can be treated symptomatically during the early stages, whereas for end stages total joint and heart valve replacements may be required [1–3], but no specific cure exists at the moment, although a phase II clinical trial with nitisinone is in progress. Thanks to the use of our in vitro, cell and tissue AKU models [4–10], we recently provided experimental evidence that AKU is a secondary serum amyloid A (SAA)-based amyloidosis [8]. A co-localization of ochronotic pigment and SAA-amyloid was also reported [8]. In the present work, using Congo Red (CR) birefringence and immunofluorescence, we report for the first time the presence of SAA-amyloidosis in the stenotic aortic valve of an AKU patient. Light microscopy revealed a co-localization of ochronotic pigment and SAA-amyloid, tissue calcification, lipid oxidation, inflammation, and tissue degeneration. Alkaptonuric specimen was obtained from a patient (Table 1) who underwent biologic aortic valve replacement. AKU patient had been previously diagnosed for secondary amyloidosis as SAA-amyloid had been detected in her cartilage and synovia [8]. Echocardiogram revealed severe aortic stenosis with an aortic transvalvular mean gradient of 53 mm Hg, a peak transvalvular gradient of 89, a peak of systolic velocity (m/s) of 4.7 and valve area of 0.82 cm2 and an AVA indexed for body weight of 0.52 cm2. Inter-ventricular septal thickness was 12 mm. Left ventricular systolic function was preserved. In view of the severe aortic stenosis the patient underwent elective aortic valve replacement. The postoperative course was uneventful and the patient recovered well from surgery at one-year follow-up. Table 1 Clinical history of the patient. After surgery AKU aortic valve sections were stained with eosin/hematoxylin, CR stained for amyloid detection or used for immunofluorescence staining with anti-SAA and anti-4-hydroxy-2-nonenal (4-HNE) antibodies. At a glance, the aortic valve revealed massive deposits of ochronotic pigment with calcification in the cusps (Fig. 1A). Sclerotic change in the cusps, and shrinkage of the non-coronary cusp, impeding normal coaptation of the aortic valve, associated with diffuse ochronotic deposition, appeared to be the causative lesions of the need for aortic valve replacement. Ochronosis was associated with areas of valvular calcification. Light microscopy examinations consistently showed severe calcification involving surface endothelium (Fig. 1B). CR staining detected the presence of diffuse amyloid, mainly located in densely sclerotic and poorly vascularised scar tissue and co-localization of amyloid and ochronotic pigment was visible in close proximity to calcific deposits (Fig. 1C). Co-localization of SAA deposition with ochronotic pigmentation was detected (Fig. 1D). Major products of lipid peroxidation (LPO) were detected in AKU valve sections incubated with anti-4-HNE antibody (Fig. 1E). LPO were perfectly superimposing to pigmented areas, suggesting the association of intraleaflet ochronosis and oxidative stress (Fig. 1E). AKU valve also contained macrophages in the subendothelial layer of the fibrosa, in the vicinity of ochronotic deposits, and along the lamina elastic (Fig. 1B). An evident co-localization between oxidized lipids, inflammation and ochronosis suggested that LPO might play a role in the disease process. Fig. 1 A) Macroscopic aspect of AKU aortic valve showing a rough surface of the leaflets and ochronotic pigmentation on the surface. A thickening of the central areas of the cusps with nodule-like structures is visible (arrow). B) Light microscopy (hematoxylin ... Our present investigation confirmed the hitherto unsuspected existence of AKU-linked secondary amyloidosis, involving also the cardiac district. The present case report is therefore a new rare case of SAA amyloidosis in the heart. The striking co-localization of pigment and amyloid suggested that HGA might be involved in amyloid deposition in the heart. In conclusion, the emerging view in this AKU case is that valve calcification represents a meeting of lipid oxidation with chronic inflammation and amyloid deposition. In AKU cases, surgical replacement of aortic valves results in significant improvement. Physicians and surgeons should be aware of multiple system involvement in this disorder, as early recognition and appropriate treatment may significantly improve the quality of life in AKU patients.

Alkaptonuria (AKU) is a rare genetic disease associated with the accumulation of homogentisic acid (HGA) and its oxidized/polymerized products which leads to the deposition of melanin-like pigments (ochronosis) in connective tissues. Although numerous case reports have described ochronosis in joints, little is known on the molecular mechanisms leading to such a phenomenon. For this reason, we characterized biochemically chondrocytes isolated from the ochronotic cartilage of AKU patients. Based on the macroscopic appearance of the ochronotic cartilage, two sub-populations were identified: cells coming from the black portion of the cartilage were referred to as “black” AKU chondrocytes, while those coming from the white portion were referred to as “white” AKU chondrocytes. Notably, both AKU chondrocytic types were characterized by increased apoptosis, NO release, and levels of pro-inflammatory cytokines. Transmission electron microscopy also revealed that intracellular ochronotic pigment deposition was common to both “white” and “black” AKU cells. We then undertook a proteomic and redox-proteomic analysis of AKU chondrocytes which revealed profound alterations in the levels of proteins involved in cell defence, protein folding, and cell organization. An increased post-translational oxidation of proteins, which also involved high molecular weight protein aggregates, was found to be particularly relevant in “black” AKU chondrocytes. J. Cell. Physiol. 227: 3333–3343, 2012. © 2011 Wiley Periodicals, Inc.

"\"Il recettore degli androgeni controlla la morfogenesi, la gametogenesi nonché la crescita e lo sviluppo della ghiandola prostatica. Esso è implicato nella progressione del tumore prostatico. Tali evidenze suggeriscono che il recettore degli androgeni controlla la migrazione cellulare. I meccanismi molecolari con cui avviene tale controllo sono ancora oggi dibattuti.. Il manoscritto dimostra che il classico recettore degli androgeni espresso in cellule mesenchimali, normali o trasformate, interagisce nel compartimento extranucleare con la filmina A e che l'aggiunta di androgeni stabilizza tale legame ed induce l'assemblaggio di un complesso ternario formato da AR\\\/filamina A ed integrina beta 1. Questo complesso da un lato attiva Rac, modificando così' la velocità di migrazione delle cellule, dall'altro induce l'attivazione della chinasi FAK, modulando in tal modo l'adesione cellulare. Il manoscritto descrive per la prima volta il meccanismo molecolare con gli androgeni promuovono la migrazione e l'invalidità di cellule bersaglio attraverso l'attivazione di vie rapide, non genomiche.\"" "\"Androgen receptor (AR) controls male morphogenesis, gametogenesis and prostate growth as well as development of prostate cancer. These findings support a role for AR in cell migration and invasiveness. However, the molecular mechanism involved in AR-mediated cell migration still remains elusive.. . Methodology\\\/Principal Findings. Mouse embryo NIH3T3 fibroblasts and highly metastatic human fibrosarcoma HT1080 cells harbor low levels of transcriptionally incompetent AR. We now report that, through extra nuclear action, AR triggers migration of both cell types upon stimulation with physiological concentrations of the androgen R1881. We analyzed the initial events leading to androgen-induced cell migration and observed that challenging NIH3T3 cells with 10 nM R1881 rapidly induces interaction of AR with filamin A (FlnA) at cytoskeleton. AR\\\/FlnA complex recruits integrin beta 1, thus activating its dependent cascade. Silencing of AR, FlnA and integrin beta 1 shows that this ternary complex controls focal adhesion kinase (FAK), paxillin and Rac, thereby driving cell migration. FAK-null fibroblasts migrate poorly and Rac inhibition by EHT impairs motility of androgen-treated NIH3T3 cells. Interestingly, FAK and Rac activation by androgens are independent of each other. Findings in human fibrosarcoma HT1080 cells strengthen the role of Rac in androgen signaling. The Rac inhibitor significantly impairs androgen-induced migration in these cells. A mutant AR, deleted of the sequence interacting with FlnA, fails to mediate FAK activation and paxillin tyrosine phosphorylation in androgen-stimulated cells, further reinforcing the role of AR\\\/FlnA interaction in androgen-mediated motility.. . Conclusions\\\/Significance. The present report, for the first time, indicates that the extra nuclear AR\\\/FlnA\\\/integrin beta 1 complex is the key by which androgen activates signaling leading to cell migration. Assembly of this ternary complex may control organ development and prostate cancer metastasis.\""

The yeast Saccharomyces cerevisiae, besides being an eukaryotic cell model, plays a fundamental role in the production of fermented foods. In the winemaking industry, yeast cell walls may be involved in numerous processes and contribute substantially to the final chemical and sensorial profiles of wines. Nonetheless, apart from mannoproteins, little is known on the protein components of the yeast cell wall and their changes during the fermentation of must into wine. In this work, we performed a dynamic analysis of the cell surface proteome (surfome) of an autochthonous wine yeast strain (previously selected as a wine fermentation starter) by shaving intact cells with trypsin and identifying tryptic peptides by means of nLC-ESI-LIT-MS/MS. Out of the 42 identified proteins, 16 and 14 were found to be specifically expressed in wine yeast surfome at the beginning and at the end of fermentation, respectively. The molecular functions of these specifically expressed proteins might help in explaining their roles in the cell wall as a response to the alcoholic fermentation-related stresses. Additionally, we provided the identification of 20 new potential cell wall related proteins. Globally, our results might provide new useful data for the selection and characterization of yeast strains to be used in the winemaking industry.

Alkaptonuria (AKU) is a rare genetic disease associated with deficient homogentisate 1,2-dioxygenase activity in the liver. This leads to the accumulation of homogentisic acid (HGA) and its oxidized/polymerized products in connective tissues, which in turn become characterized by the presence of melanin-like pigments (ochronosis). Since at present, further studies are necessary to support the use of drugs for the treatment of AKU, we investigated the effects of various anti-oxidants in counteracting melanin-like pigmentation and oxidative stress related to HGA and its metabolites.We set up an in vitro model using human serum treated with 0.33 mM HGA and tested the anti-oxidants ascorbic acid, N-acetylcysteine, phytic acid (PHY), taurine (TAU), ferulic acid (FER) and lipoic acid (LIP) for their ability to prevent or delay the production of melanin-like pigments, as well as to reduce oxidative post-translational modifications of proteins. Monitoring of intrinsic fluorescence of HGA-induced melanin-like pigments was used to evaluate the efficacy of compounds.Our model allowed us to prove efficacy especially for PHY, TAU, LIP and FER in counteracting the production of HGA-induced melanin-like pigments and protein oxidation induced by HGA and its metabolites.Our model allows the opening of new anti-oxidant therapeutic strategies to treat alkaptonuric ochronosis.

We report on a de novo submicroscopic deletion of 20q13.33 identified by subtelomeric fluorescence in situ hybridization (FISH) in a 4-year-old girl with learning difficulties, hyperlaxity and strabismus, but without obvious dysmorphic features. Further investigations by array-based comparative genomic hybridization (array-CGH) and FISH analysis allowed us to delineate the smallest reported subterminal deletion of chromosome 20q, spanning a 1.1-1.6 Mb with a breakpoint localized between BAC RP5-887L7 and RP11-261N11. The genes CHRNA4 and KCNQ2 implicated in autosomal dominant epilepsy are included in the deletion interval. Subterminal 20q deletions as found in the present patient have, to our knowledge, only been reported in three patients. We review the clinical and behavioral phenotype of such "pure" subterminal 20q deletions.

A critical region exists at 5p13 for the phenotype associated with duplication 5p. Two unrelated Polynesian children are reported with supernumerary marker chromosomes (SMCs) 5. This brings to seven the total of reported SMCs derived from chromosome 5. The phenotype is clear, and the level of mosaicism of the marker chromosomes is contributory to the clinical severity.