Alpha sarcoglycan is required for FGF-dependent myogenic progenitor cell proliferation in vitro and in vivo

Mice deficient in α-sarcoglycan (Sgca-null mice) develop progressive muscular dystrophy and serve as a model for human limb girdle muscular dystrophy type 2D. Sgca-null mice suffer a more severe myopathy than that of mdx mice, the model for Duchenne muscular dystrophy. This is the opposite of what is observed in humans and the reason for this is unknown. In an attempt to understand the cellular basis of this severe muscular dystrophy, we isolated clonal populations of myogenic progenitor cells (MPCs), the resident postnatal muscle progenitors of dystrophic and wild-type mice. MPCs from Sgca-null mice generated much smaller clones than MPCs from wild-type or mdx dystrophic mice. Impaired proliferation of Sgca-null myogenic precursors was confirmed by single fiber analysis and this difference correlated with Sgca expression during MPC proliferation. In the absence of dystrophin and associated proteins, which are only expressed after differentiation, SGCA complexes with and stabilizes FGFR1. Deficiency of Sgca leads to an absence of FGFR1 expression at the membrane and impaired MPC proliferation in response to bFGF. The low proliferation rate of Sgca-null MPCs was rescued by transduction with Sgca-expressing lentiviral vectors. When transplanted into dystrophic muscle, Sgca-null MPCs exhibited reduced engraftment. The reduced proliferative ability of Sgca-null MPCs explains, at least in part, the severity of this muscular dystrophy and also why wild-type donor progenitor cells engraft efficiently and consequently ameliorate disease.     

The nucleoporin Seh1 forms a complex with Mio and serves an essential tissue-specific function in Drosophila oogenesis

The nuclear pore complex (NPC) mediates the transport of macromolecules between the nucleus and cytoplasm. Recent evidence indicates that structural nucleoporins, the building blocks of the NPC, have a variety of unanticipated cellular functions. Here, we report an unexpected tissue-specific requirement for the structural nucleoporin Seh1 during Drosophila oogenesis. Seh1 is a component of the Nup107-160 complex, the major structural subcomplex of the NPC. We demonstrate that Seh1 associates with the product of the missing oocyte (mio) gene. In Drosophila, mio regulates nuclear architecture and meiotic progression in early ovarian cysts. Like mio, seh1 has a crucial germline function during oogenesis. In both mio and seh1 mutant ovaries, a fraction of oocytes fail to maintain the meiotic cycle and develop as pseudo-nurse cells. Moreover, the accumulation of Mio protein is greatly diminished in the seh1 mutant background. Surprisingly, our characterization of a seh1 null allele indicates that, although required in the female germline, seh1 is dispensable for the development of somatic tissues. Our work represents the first examination of seh1 function within the context of a multicellular organism. In summary, our studies demonstrate that Mio is a novel interacting partner of the conserved nucleoporin Seh1 and add to the growing body of evidence that structural nucleoporins can have novel tissue-specific roles.     

Nuclear factor erythroid 2-related factor-2 activity controls 4-hydroxynonenal metabolism and activity in prostate cancer cells

4-Hydroxynonenal (HNE) is an end product of lipoperoxidation with antiproliferative and proapoptotic properties in various tumors. Here we report a greater sensitivity to HNE in PC3 and LNCaP cells compared to DU145 cells. In contrast to PC3 and LNCaP cells, HNE-treated DU145 cells showed a smaller reduction in growth and did not undergo apoptosis. In DU145 cells, HNE did not induce ROS production and DNA damage and generated a lower amount of HNE-protein adducts. DU145 cells had a greater GSH and GST A4 content and GSH/GST-mediated HNE detoxification. Nuclear factor erythroid 2-related factor-2 (Nrf2) is a regulator of the antioxidant response. Nrf2 protein content and nuclear accumulation were higher in DU145 cells compared to PC3 and LNCaP cells, whereas the expression of KEAP1, the main negative regulator of Nrf2 activity, was lower. Inhibition of Nrf2 expression with specific siRNA resulted in a reduction in GST A4 expression and GS-HNE formation, indicating that Nrf2 controls HNE metabolism. In addition, Nrf2 knockdown sensitized DU145 cells to HNE-mediated antiproliferative and proapoptotic activity. In conclusion, we demonstrated that increased Nrf2 activity resulted in a reduction in HNE sensitivity in prostate cancer cells, suggesting a potential mechanism of resistance to pro-oxidant therapy.     

Discovery of potent nucleotide-mimicking competitive inhibitors of hepatitis C virus NS3 helicase

Among the enzymes involved in the life cycle of HCV, the non-structural protein NS3, with its double function of protease and NTPase/helicase, is essential for the virus replication. Exploiting our previous knowledge in the development of nucleotide-mimicking NS3 helicase (NS3h) inhibitors endowed with key structural and electronic features necessary for an optimal ligand-enzyme interaction, we developed the tetrahydroacridinyl derivative 3a as the most potent NS3h competitive inhibitor reported to date (HCV NS3h K(i)=20 nM).     

Synthesis and in vitro antimicrobial activities of new (cyano-NNO-azoxy)pyrazole derivatives

The antibacterial and antifungal activity of a series of products, in which the 1,5-dimethyl-4-(cyano-NNO-azoxy)pyrazol-3-yl and 1,3-dimethyl-4-(cyano-NNO-azoxy)pyrazol-5-yl moieties were linked to pyridine, pyrazole, isoxazole, thiophene and the furan ring, were examined. No molecule displayed activity against the Gram-negative bacteria tested. Conversely, some compounds displayed activity against two Staphylococcus aureus strains, including the methicillin resistant strain. All compounds displayed interesting antifungal activity, the most active compound of the series being the thiophene derivative 7a. This compound’s activity against Candidakrusei and Candidaglabrata (MIC = 0.25 and 0.5 μg/mL, respectively), two fungal species resistant to azoles, is noteworthy. The presence of the cyano function appeared essential for activity.     

Synthesis of novel antimitotic agents based on 2-amino-3-aroyl-5-(hetero)arylethynyl thiophene derivatives

Microtubules are dynamic structures that play a crucial role in cellular division and are recognized as an important target for cancer therapy. In search of new compounds with strong antiproliferative activity and simple molecular structure, a new series of 2-amino-3-(3′,4′,5′-trimethoxybenzoyl)-5-(hetero)aryl ethynyl thiophene derivatives was prepared by the Sonogashira coupling reaction of the corresponding 5-bromothiophenes with several (hetero)aryl acetylenes. When these compounds were analyzed in vitro for their inhibition of cell proliferation, the 2- and 3-thiophenyl acetylene derivatives were the most powerful compounds, both of which exerted cytostatic effects at submicromolar concentrations. In contrast, the presence of a more flexible ethyl chain between the (hetero)aryl and the 5-position of the thiophene ring resulted in significant reduction in activity relative to the 5-(hetero)aryl acetylene substituted derivatives. The effects of a selected series of compounds on cell cycle progression correlated well with their strong antiproliferative activity and inhibition of tubulin polymerization. We found that the antiproliferative effects of the most active compounds were associated with increase of the proportion of cells in the G₂/M and sub-G₁ phases of the cell cycle.     

Probing the interactions of an acyl carrier protein domain from the 6-deoxyerythronolide B synthase

The assembly‐line architecture of polyketide synthases (PKSs) provides an opportunity to rationally reprogram polyketide biosynthetic pathways to produce novel antibiotics. A fundamental challenge toward this goal is to identify the factors that control the unidirectional channeling of reactive biosynthetic intermediates through these enzymatic assembly lines. Within the catalytic cycle of every PKS module, the acyl carrier protein (ACP) first collaborates with the ketosynthase (KS) domain of the paired subunit in its own homodimeric module so as to elongate the growing polyketide chain and then with the KS domain of the next module to translocate the newly elongated polyketide chain. Using NMR spectroscopy, we investigated the features of a structurally characterized ACP domain of the 6‐deoxyerythronolide B synthase that contribute to its association with its KS translocation partner. Not only were we able to visualize selective protein–protein interactions between the two partners, but also we detected a significant influence of the acyl chain substrate on this interaction. A novel reagent, CF₃‐S‐ACP, was developed as a ¹⁹F NMR spectroscopic probe of protein–protein interactions. The implications of our findings for understanding intermodular chain translocation are discussed.     

Synthesis and biological activity of a novel class nicotinic acetylcholine receptors (nAChRs) ligands structurally related to anatoxin-a

The introduction of the isoxazole ring as bioisosteric replacement of the acetyl group of anatoxin-a led to a new series of derivatives binding to nicotinic acetylcholine receptors. Bulkier substitutions than methyl at the 3 position of isoxazole were shown to be detrimental for the activity. The binding potency of the most interesting compounds with α1, α7 and α3β4 receptor subtypes, was, anyway, only at micromolar level. Moreover, differently from known derivatives with pyridine, isoxazole condensed to azabicyclo ring led to no activity.     

Synthesis and biological profile of new 1,2,3,4-tetrahydroisoquinolines as selective carbonic anhydrase inhibitors

In a previous paper we identified several 1-aryl-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-2-sulfonamides that displayed inhibitory effects toward selected carbonic anhydrase isozymes at micromolar concentration. In order to deepen the structure-activity relationships (SARs) and identify novel compounds with improved activity, we synthesized a series of monomethoxy analogues of the previously investigated dimethoxy derivatives. The evaluation of biological profile has been focused on in vitro effects against several CA isoforms. The new monomethoxy derivatives showed higher hCA inhibitory effects against several isoforms compared to the dimethoxy analogues. Particularly, some of these compounds (e.g., 1b and 1h) showed low nanomolar K(I) values and excellent selectivity for hCA IX and hCA XIV versus hCA I and II inhibition.     

Enriched sera protein profiling for detection of non-small cell lung cancer biomarkers

Background

Non Small Cell Lung Cancer (NSCLC) is the major cause of cancer related-death. Many patients receive diagnosis at advanced stage leading to a poor prognosis. At present, no satisfactory screening tests are available in clinical practice and the discovery and validation of new biomarkers is mandatory. Surface Enhanced Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (SELDI-ToF-MS) is a recent high-throughput technique used to detect new tumour markers. In this study we performed SELDI-ToF-MS analysis on serum samples treated with the ProteoMiner? kit, a combinatorial library of hexapeptide ligands coupled to beads, to reduce the wide dynamic range of protein concentration in the sample. Serum from 44 NSCLC patients and 19 healthy controls were analyzed with IMAC30-Cu and H50 ProteinChip Arrays.

Results

Comparing SELDI-ToF-MS protein profiles of NSCLC patients and healthy controls, 28 protein peaks were found significantly different (p < 0.05), and were used as predictors to build decision classification trees. This statistical analysis selected 10 protein peaks in the low-mass range (2-24 kDa) and 6 in the high-mass range (40-80 kDa). The classification models for the low-mass range had a sensitivity and specificity of 70.45% (31/44) and 68.42% (13/19) for IMAC30-Cu, and 72.73% (32/44) and 73.68% (14/19) for H50 ProteinChip Arrays.

Conclusions

These preliminary results suggest that SELDI-ToF-MS protein profiling of serum samples pretreated with ProteoMiner? can improve the discovery of protein peaks differentially expressed between NSCLC patients and healthy subjects, useful to build classification algorithms with high sensitivity and specificity. However, identification of the significantly different protein peaks needs further study in order to provide a better understanding of the biological nature of these potential biomarkers and their role in the underlying disease process.