Search

From the first implementation in its purely hydrodynamic configuration, to the last configuration under the Coupled Ocean-Atmosphere-Wave-Sediment Transport (COAWST) system, several specific modelling applications of the Regional Ocean Modelling Systems (ROMS, www.myroms.org) have been put forward within the Adriatic Sea (Italy) region. Covering now a wide range of spatial and temporal scales, they developed in a growing number of fields supporting Integrated Coastal Zone Management (ICZM) and Marine Spatial Planning (MSP) activities in this semi-enclosed sea of paramount importance including the Gulf of Venice. Presently, a ROMS operational implementation provides every day hydrodynamic and sea level 3-days forecasts, while a second one models the most relevant biogeochemical properties, and a third one (two-way coupled with the Simulating Waves Nearshore (SWAN) model) deals with extreme waves forecast. Such operational models provide support to civil and environmental protection activities (e.g., driving sub-models for oil-spill dispersion, storm surge, coastal morphodynamic changes during storms, saline wedge intrusion along Po River), in a growing context of stake-holders at regional, national and international level. Besides, ROMS and COAWST research based activities are also carried out, mostly aiming at investigating sediment transport, eggs and larvae dispersion, hypoxic events in the basin; through successive nesting very high resolutions nearshore the Italian coast can be reached, allowing to simulate river mouth environments and artificial reefs. Resulting outputs, written in NetCDF CF compliant format, are delivered via THREDDS Data Server to a growing number of users around the world., Comment: pre-print of paper to be completed and submitted. Revised on Feb 2014

It is well know that the ocean processes exert a great influence on global climate as well as affect the local climate of coastal areas (Russo et al., 2002). Within the Mediterranean region (see Fig. 1a), the presence of the Adriatic Sea influences the atmospheric properties of the surrounding regions over long and short time-scales, and has obviously a relevant influence on human activities and ecosystems (Boldrin et al., 2009)., http://digitalcommons.usu.edu/modern_climatology/1006/thumbnail.jpg

RNase A dimers have been recently found to be endowed with some of the special, i.e., non-catalytic biol. activities of RNases, such as antitumor and aspermatogenic activities. These activities have been so far attributed to RNases which can escape the neutralizing action of the cytosolic RNase inhibitor (cRI). However, when the interactions of the two cytotoxic RNase A dimers with cRI were investigated in a quant. fashion and at the mol. level, the dimers were found to bind cRI with high affinity and to form tight complexes.

Coastal areas host a large fraction of the world's population and are exposed to natural extreme events, which are a serious threat to human life, as well as to economies. For this reason, sea storms are increasingly the object of studies, and the design of traditional coastal defenses is being carried out in conjunction with modeling analyses. Relying on numerical simulations performed by means of an innovative shallow-water hydro-morphodynamic model, the present work explores the overall response of a protected beach to sea storms. Numerical tests evaluate the effects of sea states extracted from realistic sea storms having different spectral characteristics, as well as the influence on beach morphology of positioning shore-parallel, impermeable, submerged breakwaters. Simulation results revealed that, while erosion/accretion patterns depend weakly on the different sea state conditions, the morphodynamics induced around the barriers is strongly influenced by the breakwaters' positioning. More specifically, at least for the forcing here analyzed, bed variations were shown to increase when the structures are progressively located offshore; on the other hand, the swash zone morphology seems to be only weakly influenced by the positioning of the breakwaters. We also observed that for an increasing extension of the volume over which dissipative breaking mechanisms occur, a decreasing inshore erosion is accompanied by an equally fast decrease of offshore erosion. Analysis of the vorticity fields shows that breakwaters placed far from the shoreline induce an evolution of the vortices generated by breaking waves rather different from the one due to breakwaters placed closer to the shoreline (which can induce seaward flows through the gap, like rip currents).

Given the increasing interest in bioactive dietary components that can modulate gene expression enhancing human health, three metabolites isolated from hemp seeds—cannabidiolic acid, N-trans-caffeoyltyramine, and cannabisin B—were examined for their ability to change the expression levels of microRNAs in human neural cells. To this end, cultured SH-SY5Y cells were treated with the three compounds and their microRNA content was characterized by next-generation small RNA sequencing. As a result, 31 microRNAs underwent major expression changes, being at least doubled or halved by the treatments. A computational analysis of the biological pathways affected by these microRNAs then showed that some are implicated in neural functions, such as axon guidance, hippocampal signaling, and neurotrophin signaling. Of these, miR-708-5p, miR-181a-5p, miR-190a-5p, miR-199a-5p, and miR-143-3p are known to be involved in Alzheimer’s disease and their expression changes are expected to ameliorate neural function. Overall, these results provide new insights into the mechanism of action of hemp seed metabolites and encourage further studies to gain a better understanding of their biological effects on the central nervous system.

// Nicola Coppola 1 , Giorgio de Stefano 2 , Marta Panella 3 , Lorenzo Onorato 1 , Valentina Iodice 2 , Carmine Minichini 1 , Nicola Mosca 3 , Luisa Desiato 3 , Nunzia Farella 2 , Mario Starace 1 , Giulia Liorre 2 , Nicoletta Potenza 3 , Evangelista Sagnelli 1 , Aniello Russo 3 1 Department of Mental Health and Public Medicine, Second University of Naples, Naples, Italy 2 IX Interventional Ultrasound Unit for Infectious Diseases, AORN dei Colli, P.O. Cotugno, Naples, Italy 3 Department of Environmental, Biological and Pharmaceutical Sciences and Technologies, Second University of Naples, Naples, Italy Correspondence to: Aniello Russo, email: aniello.russo@unina2.it Keywords: microRNA, gene regulation, cell proliferation, tumor, hepatocellular carcinoma Received: November 08, 2016 Accepted: February 07, 2017 Published: February 28, 2017 ABSTRACT Human microRNA-125a-5p (miR-125a) is expressed in most tissues where it downregulates the expression of membrane receptors or intracellular transductors of mitogenic signals, thus limiting cell proliferation. Expression of this miRNA generally increases with cell differentiation whereas it is downregulated in several types of tumors, such as breast, lung, ovarian, gastric, colon, and cervical cancers, neuroblastoma, medulloblastoma, glioblastoma, and retinoblastoma. In this study, we focused on hepatocellular carcinoma and used real-time quantitative PCR to measure miR-125a expression in 55 tumor biopsies and in matched adjacent non-tumor liver tissues. This analysis showed a downregulation of miR-125a in 80 % of patients, with a mean decrease of 4.7-fold. Comparison of miRNA downregulation with clinicopathological parameters of patients didn’t yield significant correlations except for serum bilirubin. We then evaluated the expression of known targets of miR-125a and found that sirtuin-7, matrix metalloproteinase-11, and c-Raf were up-regulated in tumor tissue by 2.2-, 3-, and 1.7-fold, respectively. Overall, these data support a tumor suppressor role for miR-125a and encourage further studies aimed at the comprehension of the molecular mechanisms governing its expression, eventually leading to treatments to restore its expression in tumor cells.

The discovery of bioactive compounds from natural sources entails an extremely lengthy process due to the timescale and complexity of traditional methodologies. In our study, we used a rapid NMR based metabolomic approach as tool to identify secondary metabolites with anti-proliferative activity against a panel of human colorectal cancer cell lines with different mutation profiles. For this purpose, fourteen Fabaceae species of Mediterranean vegetation were investigated using a double screening method: 1H NMR profiling enabled the identification of the main compounds present in the mixtures, whilst parallel biological assays allowed the selection of two plant extracts based on their strong anti-proliferative properties. Using high-resolution 2D NMR spectroscopy, putative active constituents were identified in the mixture and isolated by performing a bio-guided fractionation of the selected plant extracts. As a result, we found two active principles: a cycloartane glycoside and protodioscin derivative. Interestingly, these metabolites displayed a preferential anti-proliferative effect on colon cancer cell lines with an intrinsic resistance to anti-EGFR therapies. Our work provides an NMR-based metabolomic approach as a powerful and efficient tool to discover natural products with anticancer activities circumventing time-consuming procedures.

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system, characterized by chronic inflammation, demyelination and scarring as well as a broad spectrum of signs and symptoms. MicroRNA plays pivotal roles in cellular and developmental processes by regulating gene expression at the post-transcriptional level. Increasing evidence suggests the involvement of microRNAs in the pathogenesis of neurodegenerative diseases, including MS. We have already found that the expression of a specific miRNA, hsa-mir-26a-5p (miR-26a), changed during INF-β treatment in responder Relapsing-Remitting MS patients. Functional annotations of mir-26a targets revealed that a number of genes were implicated in Glutamate Receptor Signaling pathway, which is notoriously altered in neurodegenerative diseases as MS. In this study, the different potential targets were subjected to a validation test based on luciferase reporter constructs transfected in an oligodendroglial cell line. In this functional screening, miR-26a was able to interact with SLC1A1 3' UTR suppressing the reporter activity. Transfection of a miR-26a mimic was then shown to decrease the endogenous SLC1A1 mRNA. Afterward, we have evaluated in blood platelets from interferon-β treated Multiple Sclerosis patients the expression of miR-26a and SLC1A1, finding not only their converse expression, but also a responsiveness to interferon-β therapy. Overall, these data suggest that mir-26a and SLC1A1 may play a role in the MS pathogenesis, and may be potential targets for the development of new biomarkers and/or therapeutic tools.

The hepatitis B virus (HBV) is a widespread human pathogen and chronic HBV infection is a major risk factor for hepatocellular carcinoma (HCC). Some cellular microRNAs are emerging as important regulators of virus-host interaction, indirectly or directly modulating HBV replication and pathogenesis. miR-125a binds the viral transcript encoding the surface antigen and interferes with its expression, thus inhibiting viral replication. Intriguingly, liver miR-125a expression has been found increased in patients with high levels of hepatic HBV-DNA. The present study investigates the mechanism by which liver exposure to HBV induces the expression of miR-125a. The analyses were first performed on liver biopsies from HBV patients, showing that the expression of the viral transactivator X protein (HBx) paralleled the increase of miR-125a expression. Then, transfection of HCC cell lines with an HBx-expressing vector showed a substantial increase of miR-125a expression. Overall, the available data depict a self-inhibitory feedback loop in which HBV, through HBx, increases the expression of miR-125a, that in turn interferes with expression of HBV surface antigen, thus repressing viral replication. © 2014 Elsevier Inc. All rights reserved. The hepatitis B virus (HBV) is a widespread human pathogen and chronic HBV infection is a major risk factor for hepatocellular carcinoma (HCC). Some cellular microRNAs are emerging as important regulators of virus-host interaction, indirectly or directly modulating HBV replication and pathogenesis. miR-125a binds the viral transcript encoding the surface antigen and interferes with its expression, thus inhibiting viral replication. Intriguingly, liver miR-125a expression has been found increased in patients with high levels of hepatic HBV-DNA. The present study investigates the mechanism by which liver exposure to HBV induces the expression of miR-125a. The analyses were first performed on liver biopsies from HBV patients, showing that the expression of the viral transactivator X protein (HBx) paralleled the increase of miR-125a expression. Then, transfection of HCC cell lines with an HBx-expressing vector showed a substantial increase of miR-125a expression. Overall, the available data depict a self-inhibitory feedback loop in which HBV, through HBx, increases the expression of miR-125a, that in turn interferes with expression of HBV surface antigen, thus repressing viral replication. © 2014 Elsevier Inc. All rights reserved.

This work reports the results of the application of a practical method to determine the in vivo dose at the isocenter point, D(iso), of brain thorax and pelvic treatments using a transit signal S(t). The use of a stable detector for the measurement of the signal S(t) (obtained by the x-ray beam transmitted through the patient) reduces many of the disadvantages associated with the use of solid-state detectors positioned on the patient as their periodic recalibration, and their positioning is time consuming. The method makes use of a set of correlation functions, obtained by the ratio between S(t) and the mid-plane dose value, D(m), in standard water-equivalent phantoms, both determined along the beam central axis. The in vivo measurement of D(iso) required the determination of the water-equivalent thickness of the patient along the beam central axis by the treatment planning system that uses the electron densities supplied by calibrated Hounsfield numbers of the computed tomography scanner. This way it is, therefore, possible to compare D(iso) with the stated doses, D(iso,TPS), generally used by the treatment planning system for the determination of the monitor units. The method was applied in five Italian centers that used beams of 6 MV, 10 MV, 15 MV x-rays and (60)Co gamma-rays. In particular, in four centers small ion-chambers were positioned below the patient and used for the S(t) measurement. In only one center, the S(t) signals were obtained directly by the central pixels of an EPID (electronic portal imaging device) equipped with commercial software that enabled its use as a stable detector. In the four centers where an ion-chamber was positioned on the EPID, 60 pelvic treatments were followed for two fields, an anterior-posterior or a posterior-anterior irradiation and a lateral-lateral irradiation. Moreover, ten brain tumors were checked for a lateral-lateral irradiation, and five lung tumors carried out with three irradiations with different gantry angles were followed. One center used the EPID as a detector for the S(t) measurement and five pelvic treatments with six fields (many with oblique incidence) were followed. These last results are reported together with those obtained in the same center during a pilot study on ten pelvic treatments carried out by four orthogonal fields. The tolerance/action levels for every radiotherapy fraction were 4% and 5% for the brain (symmetric inhomogeneities) and thorax/pelvic (asymmetric inhomogeneities) irradiations, respectively. This way the variations between the total measured and prescribed doses at the isocenter point in five fractions were well within 2% for the brain treatment, and 4% for thorax/pelvic treatments. Only 4 out of 90 patients needed new replanning, 2 patients of which needed a new CT scan.

Defensins, RNases and cytokines are present at mucosal barriers, main ports of HIV entry, and are potential mediators of the resistant phenotype exhibited by HIV-1-exposed seronegative individuals (HESN) during sexual exposure. We aimed to determine the role of soluble factors in natural resistance to HIV-1 infection. Vaginal/endocervical/oral mucosal samples were taken from 60 HESN, 60 seropositive (SP) and 61 healthy controls (HC). Human neutrophil peptide 1 (hNP-1), human beta defensin (hBD) 2 and 3, RNases, MIP-1β and RANTES mRNA transcripts were quantified by qPCR and in vitro single-round, recombinant-based viral infectivity assay was used to evaluate the anti-HIV-1 activity of hBDs and RNases. HESN expressed significantly higher levels of hNP-1, hBDs mRNA in oral mucosa compared to HC (P < 0.05). In genital mucosa, significantly higher mRNA levels of MIP-1β, RANTES and RNases were found in HESN compared to HC (P < 0.05). HBDs and RNases inhibit HIV-1 replication, particularly R5 at entry, reverse transcription and nuclear import of the viral life cycle. hNP-1, hBDs, MIP-1β, RANTES and RNases, collectively could contribute to HIV-1 resistance during sexual exposure. Moreover, the inhibition of HIV-1 infection in vitro by hBDs and RNases suggests that they may be exploited as potential antiretrovirals.

Human ribonuclease-1 (hRNase-1) is an extracellular enzyme found in exocrine pancreas, blood, milk, saliva, urine and seminal plasma, which has been implicated in digestion of dietary RNA and in antiviral host defense. The enzyme is characterized by a high catalytic activity toward both single-stranded and double-stranded RNA. In this study, we explored the possibility that hRNase-1 may also be provided with a ribonuclease H activity, i.e. be able to digest the RNA component of RNA:DNA hybrids. For this purpose, we developed an accurate and sensitive real-time RNase H assay based on a fluorogenic substrate made of a 12 nt 50-fluorescein-labeled RNA hybridized to a complementary 30-quenchermodified DNA. Under physiological-like conditions, hRNase-1 was found to cleave the RNA:DNA hybrid very efficiently, as expressed by a kcat/Km of 330 000 M-1 s-1, a value that is over 180-fold higher than that obtained with the homologous bovine RNase A and only 8-fold lower than that measured with Escherichia coli RNase H. The kinetic characterization of hRNase-1 showed that its hybridase activity is maximal at neutral pH, increases with lowering ionic strength and is fully inhibited by the cytosolic RNase inhibitor. Overall, the reported data widen our knowledge of the enzymatic properties of hRNase-1 and provide new elements for the comprehension of its biological function. Human ribonuclease-1 (hRNase-1) is an extracellular enzyme found in exocrine pancreas, blood, milk, saliva, urine and seminal plasma, which has been implicated in digestion of dietary RNA and in antiviral host defense. The enzyme is characterized by a high catalytic activity toward both single-stranded and double-stranded RNA. In this study, we explored the possibility that hRNase-1 may also be provided with a ribonuclease H activity, i.e. be able to digest the RNA component of RNA:DNA hybrids. For this purpose, we developed an accurate and sensitive real-time RNase H assay based on a fluorogenic substrate made of a 12 nt 5'-fluorescein-labeled RNA hybridized to a complementary 3'-quenchermodified DNA. Under physiological-like conditions, hRNase-1 was found to cleave the RNA:DNA hybrid very efficiently, as expressed by a k(cat)/K-m of 330 000 M-1 s(-1), a value that is over 180-fold higher than that obtained with the homologous bovine RNase A and only 8-fold lower than that measured with Escherichia coli RNase H. The kinetic characterization of hRNase-1 showed that its hybridase activity is maximal at neutral pH, increases with lowering ionic strength and is fully inhibited by the cytosolic RNase inhibitor. Overall, the reported data widen our knowledge of the enzymatic properties of hRNase-1 and provide new elements for the comprehension of its biological function.

Human angiogenin (Ang) is an RNase in the pancreatic RNase superfamily that induces angiogenesis. Its catalytic activity is comparatively weak, but nonetheless critical for biological activity. The crystal structure of Ang has shown that enzymatic potency is attenuated in part by the obstructive positioning of Gln 117 within the B 1 pyrimidine binding pocket, and that the C-terminal segment of residues 117-123 must reorient for Ang to bind and cleave RNA. The native closed conformation appears to be stabilized by Gln117-Thr44 and Asp116-Ser118 hydrogen bonds, as well as hydrophobic packing of Ilel 19 and Phel20. Consistent with this view, Q117G, D116H, and I119A/F120A variants are 4-30-fold more active than Ang. Here we have determined crystal structures for these variants to examine the structural basis for the activity increases. In all three cases, the C-terminal segment remains obstructive, demonstrating that none of the residues that has been replaced is essential for maintaining the closed conformation. The Q117G structure shows no changes other than the loss of the side chain of residue 117, whereas those of D116H and I119A/F120A reveal C-terminal perturbations beyond the replacement site, suggesting that the native closed conformation has been destabilized. Thus, the interactions of Gln117 seem to be less important than those of residues 116, 119, and 120 for stabilization. In D116H, His 116 does not replicate either of the hydrogen bonds of Aspl 16 with Ser118 and instead forms a water-mediated interaction with catalytic residue His114; residues 117-121 deviate significantly from their positions in Ang. In I119A/ F120A, the segment of residues 117-123 has become highly mobile and all of the interactions thought to position Gln117 have been weakened or lost; the space occupied by Phel20 in Ang is partially filled by Arg101, which has moved several angstroms. A crystal structure was also determined for the deletion mutant des(121-123), which has 10-fold reduced activity toward large substrates. The structure is consistent with the earlier proposal that residues 121-123 form part of a peripheral substrate binding subsite, but also raises the possibility that changes in the position of another residue, Lys82, might be responsible for the decreased activity of this variant.

Molecular modeling based on the crystal structure of the complex of bovine pancreatic RNase A with the inhibitor 5'-diphosphoadenosine 3'- phosphate (ppAp) (Leonidas, D. D., Shapiro, R., Irons, L. I., Russo, N., and Acharya, K. R. (1997) Biochemistry 36, 5578-5588) was used to design new inhibitors that extend into unoccupied regions of the enzyme active site. These compounds are dinucleotides that contain an unusual 3',5'- pyrophosphate linkage and were synthesized in solution by a combined chemical and enzymatic procedure. The most potent of them, 5'-phospho-2'-deoxyuridine 3'-pyrophosphate, P' → 5'-ester with adenosine 3'-phosphate (pdUppAp), binds to RNase A with K(i) values of 27 and 220 nM at pH 5.9 and 7, respectively. These values are 6-9-fold lower than those for ppAp and 50-fold lower than that for the transition state analogue, uridine vanadate. pdUppAp has broad specificity; it is an effective inhibitor of at least two other members of the pancreatic RNase superfamily, human RNase-2 (eosinophil-derived neurotoxin) and RNase-4, which share only 36-44% sequence identity with the pancreatic enzyme. The potency of pdUppAp and the other inhibitors described here depends critically on the extended internucleotide linkage; the pyrophosphate group enhances dinucleotide binding to the three RNases by 2.1- 2.9 orders of magnitude, as compared with a monophosphate. These data give further insight into the organization of the catalytic centers of the various RNases. Moreover, the new class of inhibitors provides a useful means by which to probe the biological actions of these and other related enzymes.

Specific ligands (aptamers) for angiogenin were selected from a 72-mer oligodeoxynucleotide library consisting of 28 randomized positions flanked by two constant regions of 22 residues each. From a starting pool of approximately 10(14) molecules, 19 angiogenin-binding ligands were obtained. Among them, two oligonucleotides showed significant inhibition of the ribonucleolytic activity of angiogenin with apparent Kis of 0.65 and 0.60 micro M, respectively. One of them was shortened on the basis of its secondary structure to provide a 45-mer oligonucleotide that retained much of the inhibitory properties of the parent molecule. It inhibits both the angiogenic and cell proliferative activities of angiogenin but does not interfere with its nuclear translocation in human endothelial cells. Importantly, the inhibitor is cotranslocated to the nucleus with angiogenin in a approximately 1:1 stoichiometric ratio. These results demonstrate that the inhibition of angiogenin-induced cell proliferation and angiogenesis by the oligonucleotide is due to suppression of the ribonucleolytic activity of angiogenin, an event that occurs most likely within the cell nucleus.

Aims MicroRNAs (miRNAs) play an important role in the pathogenesis of structural alterations of the failing heart through their ability to regulate negatively the expression levels of genes that govern the process of adaptive and maladaptive cardiac remodelling.We studied whether LV reverse remodelling after CRTwas associated with changes of circulating miRNAs in patients with heart failure (HF) and dyssynchrony. Methods and results A prospective, non-randomized self-control trial was performed in 81 patients with HF eligible for CRT. At baseline, to select the HF miRNA profile, we evaluated the expression of 84 miRNAs (implicated in the pathogenesis of structural alterations of the failing heart) in three groups of patients: healthy subjects (healthy group, n = 15); patients with HF (HF group, n = 81); and patients without HF matched for age, sex, and concomitant disease with HF patients (control group, n = 60). At 12 months, the selected miRNA profile was evaluated in plasma from responder (n = 55) and non-responder HF patients (n = 26) to CRT. In the test cohort, the HF patientswere characterized by lower expression of 48 miRNAs (all P < 0.04) as compared with healthy subjects. In the validation cohort, the HF patients were characterized by lower expression of 24miRNAs(all P < 0.03) as compared with control patients. At 12 months, 55 patients (68%) were considered responders and 26 non-responders to CRT (32%). Responders showed an increase in expression of 19 miRNAs (all P < 0.03) compared with baseline expression, whereas in the non-responders we observed an increase of six miRNAs (all P < 0.05) compared with baseline expression. At follow-up, miRNAs were differentially expressed between responders and non-responders. The responders were characterized by higher expression of five miRNAs (miRNA-26b-5p, miRNA-145-5p, miRNA-92a-3p, miRNA-30e-5p, and miRNA-29a-3p; P < 0.01 for all) as compared with non-responders. Conclusions In responders, reverse remodelling is associated with favourable changes in miRNAs that regulate cardiac fibrosis, apoptosis, and hypertrophy. © The Author 2013. For permissions please email: journals.permissions@oup.com.

In the assessment of the antioxidant properties of edible plants, the widely consumed Vitis × labruscana cv. 'Isabella', known in Italy as "fragola" (strawberry) grape, was of interest. Phenol and flavonoid contents of the methanolic extracts of peel, pulp, seed, leaf, and stalk components of the plant were determined. The metabolic profile of the extracts was performed by 1D and 2D NMR. Quantitative analysis, obtained in the presence of 0.01% of internal standard trimethylsilyl propionate, evidenced the presence of catechins in both stalk and seed extracts, whereas caffeic acid and quercetin were the main metabolites of the leaf extract. Furthermore, the extracts were tested for their radical scavenging and reducing capacities by measuring their capacity to scavenge DPPH• and ABTS•+and to reduce Fe(III) and Mo(VI) salts. The antioxidant efficacy of the extracts in cell-free systems and their antiproliferative activity toward HepG2 and A549 cells were also evaluated. Seed and stalk components are able to reduce by 39.6 and 40.6%, respectively, the amount of the metabolically active HepG2 cells after only 24 h of exposure. © 2011 American Chemical Society.

Dicer and Argonaute 1-4 proteins are key components of the cytoplasmic enzyme machinery responsible for biogenesis and performance of microRNAs. To gain insight into the roles of these proteins in cell differentiation, we investigated possible changes in the expression levels of Dicer and Argonaute 1-4 genes during the differentiation of cultured neural and glial cells. The results show that the 5 genes are differentially expressed along the 2 differentiation pathways and suggest a prevalent role of Dicer and Argonaute 4 in neural cell differentiation. © 2009 International Federation for Cell Biology.

The RNase H domain of human hepatitis B virus (HBV) polymerase is an attractive molecular target for the development of new anti-HBV drugs. In this study, a synthetic gene coding for HBV RNase H was assembled from 12 oligonucleotides and expressed in Escherichia coli. The encoded protein was then recovered from inclusion bodies, purified, and refolded by a dilution-dialysis procedure in the presence of a low concentration of lauroylsarcosine (0.01%). The presence of the detergent was an absolute requirement for solubility, suggesting that the untagged RNase H might have exposed hydrophobic regions that need to be shielded from the solvent. The structural identity of the protein was confirmed by N-terminal amino acid sequence analysis and mass spectrometry. The enzymatic activity of HBV RNase H was then tested by a recently developed fluorometric assay and was found to be only slightly lower than that registered with the entire HIV-1 reverse transcriptase. Finally, a structural model of the enzyme showed that H715, R744 and K745 may be involved in substrate recognition. © 2007 Elsevier Inc. All rights reserved.

Site-directed mutagenesis of human pancreatic RNase (HP-RNase) was used as a model system for investigating the genetic events underlying the evolutionary origins of protein oligomers. HP-RNase is a monomeric enzyme with no natural tendency to oligomerize (K(d) for any dimers in solution of280 mM). Nevertheless, deletion of five amino acid residues in the loop linking the N-terminal helix of HP-RNase to the rest of the protein was found to drive polypeptide chains to fold into dimers. These dimers could not be dissociated by heating at 70 degrees C, and small amounts of monomer were detected only in highly diluted samples. Measurement of dimer and monomer concentrations under equilibrium conditions yielded a K(d) of 1.5 microM. This implies that the deletion increases the protein propensity to dimerize at least 5.2 orders of magnitude. Moreover, the HP-RNase dimers were found to be over 4.6 orders of magnitude more stable than the dimers of bovine pancreatic RNase A obtained by lyophilization from acetic acid (K(d)73 mM). Cross-linking experiments with divinyl sulfone indicated that the HP-RNase dimers are stabilized by the exchange between subunits of their N-terminal helices. This generates composite active sites, i.e., each contributed by two subunit chains, that retain full enzymatic activity. Overall, these results show that a deletion of few residues in a key region of a monomeric protein can be the primary event irreversibly leading to oligomerization of the protein through the swap of a secondary structure element between protomers.

In 1965 Fruchter and Crestfield (J. Biol. Chem. 240, 2868-3874) observed that dimeric RNase A prepared by lyophilization from acetic acid could be separated into two forms. Surprisingly, no other structural or functional differences could be detected between the two forms. In 1998 a structure for dimeric RNase A was determined by X-ray crystallography by Liu et al. (Proc. Natl. Acad. Sci. USA 95, 3437-3442). We found that the two forms of dimeric RNase A have indeed different structural and functional properties, and suggest that the dimer whose structure was investigated by Liu and coworkers may be identified with the lesser form of dimeric RNase A. Copyright (C) 2000 Federation of European Biochemical Societies.

The crystal structure of ribonuclease A (RNase A) in complex with pdUppA-3'-p [5'-phospho-2'-deoxyuridine-3'-pyrophosphate (P'-->5') adenosine 3'-phosphate] has been determined at 1.7 A resolution. This dinucleotide is the most potent low molecular weight inhibitor of RNase A reported to date (K(i) = 27 nM) and is also effective against two major nonpancreatic RNases: eosinophil-derived neurotoxin and RNase-4; in all cases, tight binding in large part derives from the unusual 3',5'-pyrophosphate internucleotide linkage [Russo, N., and Shapiro, R. (1999) J. Biol. Chem. 274, 14902-14908]. The design of pdUppA-3'-p was based on the crystal structure of RNase A complexed with 5'-diphosphoadenosine 3'-phosphate (ppA-3'-p) [Leonidas, D. D., Shapiro, R., Irons, L. I., Russo, N., and Acharya, K. R. (1997) Biochemistry 36, 5578-5588]. The adenosine of pdUppA-3'-p adopts an atypical syn conformation not observed for standard adenosine nucleotides bound to RNase A. This conformation, which allows extensive interactions with Asn 67, Gln 69, Asn 71, and His 119, is associated with the placement of the 5'-beta-phosphate of the adenylate, rather than alpha-phosphate, at the site where substrate phosphodiester bond cleavage occurs. The contacts of the deoxyuridine 5'-phosphate portion of pdUppA-3'-p appear to be responsible for the 9-fold increased affinity of this compound as compared to ppA-3'-p: the uracil base binds to Thr 45 in the same manner as previous pyrimidine inhibitors, and the terminal 5'-phosphate is positioned to form medium-range Coulombic interactions with Lys 66. The full potential benefit of these added interactions is not realized because of compensatory losses of hydrogen bonds of Lys 7 and Gln 11 with the terminal 3'-phosphate and the adenylate 5'-alpha-phosphate, which were not predicted by modeling. The results reported here have important implications for the design of improved inhibitors of RNase A and for the development of therapeutic agents to control the activities of RNase homologues such as eosinophil-derived neurotoxin and angiogenin that have roles in human pathologies.

To study in HBsAg chronic carriers the expression of liver hsa-miR-125a-5p and its correlation with liver HBV-DNA values and clinical presentation, 27 consecutive Caucasian, HBsAg/anti-HBe/HBV-DNA-positive patients who were naive to nucleos(t)ide analogues and interferon therapy and had no marker of HCV, HDV or HIV infection and no history of alcohol intake were enrolled. For each patient, liver HBV DNA and liver hsa-miR-125a-5p were quantified by real-time PCR in relation to β-globin DNA or RNU6B, respectively. Liver fibrosis and necroinflammation were graded by applying Ishak's scoring system. Liver hsa-miR-125a-5p was detected in all patients enrolled and a correlation between its concentration and liver HBV DNA was demonstrated (p103 IU/ml (p6 (p = 0.02) and those with fibrosis score >2 (p2 (p = 0.02) than in patients with a lower load. By multivariate logistic regression analysis, liver hsa-miR-125a-5p was identified as an independent predictor of disease progression: O.R. = 4.21, C.I. 95% = 1.08-16.43, p6; O.R. = 3.12, C.I. 95% = 1.17-8.27, p2. In conclusion, in HBsAg/anti-HBe-positive patients, the liver hsa-miR-125a-5p level correlated with liver and plasma HBV-DNA values and was associated to a more severe disease progression. © 2013 Coppola et al. To study in HBsAg chronic carriers the expression of liver hsa-miR-125a-5p and its correlation with liver HBV-DNA values and clinical presentation, 27 consecutive Caucasian, HBsAg/anti-HBe/HBV-DNA-positive patients who were naive to nucleos(t)ide analogues and interferon therapy and had no marker of HCV, HDV or HIV infection and no history of alcohol intake were enrolled. For each patient, liver HBV DNA and liver hsa-miR-125a-5p were quantified by real-time PCR in relation to β-globin DNA or RNU6B, respectively. Liver fibrosis and necroinflammation were graded by applying Ishak's scoring system. Liver hsa-miR-125a-5p was detected in all patients enrolled and a correlation between its concentration and liver HBV DNA was demonstrated (p103 IU/ml (p6 (p = 0.02) and those with fibrosis score >2 (p2 (p = 0.02) than in patients with a lower load. By multivariate logistic regression analysis, liver hsa-miR-125a-5p was identified as an independent predictor of disease progression: O.R. = 4.21, C.I. 95% = 1.08-16.43, p6; O.R. = 3.12, C.I. 95% = 1.17-8.27, p2. In conclusion, in HBsAg/anti-HBe-positive patients, the liver hsa-miR-125a-5p level correlated with liver and plasma HBV-DNA values and was associated to a more severe disease progression. © 2013 Coppola et al.

A PCR procedure for synthesizing genes which uses medium lenght oligonucleotides is described. The procedure is tested leading to the synthesis of the cDNA coding for HL-RNase.

Bovine seminal ribonuclease, a uniquely dimeric pancreatic-like RNase, with its dimeric structure stabilized by two intersubunit disulfides, and endowed with special, i.e. non-catalytic, actions (antitumor, immunosuppressive, antispermatogenic), was stably expressed in Chinese hamster ovary cells. The recombinant protein, secreted in the culture medium as a correctly folded dimeric enzyme, was purified to homogeneity and found to be fully active both in its catalytic and antitumor activities. © 1993.