The translation initiation functions of IF2: targets for thiostrepton inhibition

Bacterial translation initiation factor IF2 was localized on the ribosome by rRNA cleavage using free Cu(II):1,10-orthophenanthroline. The results indicated proximity of IF2 to helix 89, to the sarcin-ricin loop and to helices 43 and 44, which constitute the "L11/thiostrepton" stem-loops of 23S rRNA. These findings prompted an investigation of the L11 contribution to IF2 activity and a re-examination of the controversial issue of the effect on IF2 functions of thiostrepton, a peptide antibiotic known primarily as a powerful inhibitor of translocation. Ribosomes lacking L11 were found to have wild-type capacity to bind IF2 but a strongly reduced ability to elicit its GTPase activity. We found that thiostrepton caused a faster recycling of this factor on and off the 70S ribosomes and 50S subunits, which in turn resulted in an increased rate of the multiple turnover IF2-dependent GTPase. Although thiostrepton did not inhibit the P-site binding of fMet-tRNA, the A-site binding of the EF-Tu-GTP-Phe-tRNA or the activity of the ribosomal peptidyl transferase center (as measured by the formation of fMet-puromycin), it severely inhibited IF2-dependent initiation dipeptide formation. This inhibition can probably be traced back to a thiostrepton-induced distortion of the ribosomal-binding site of IF2, which leads to a non-productive interaction between the ribosome and the aminoacyl-tRNA substrates of the peptidyl transferase reaction. Overall, our data indicate that the translation initiation function of IF2 is as sensitive as the translocation function of EF-G to thiostrepton inhibition.     

Functionalized pyrazoles and pyrazolo[3,4-d]pyridazinones: Synthesis and evaluation of their phosphodiesterase 4 inhibitory activity

A series of pyrazoles and pyrazolo[3,4-d]pyridazinones were synthesized and evaluated for their PDE4 inhibitory activity. All the pyrazoles were found devoid of activity, whereas some of the novel pyrazolo[3,4-d]pyridazinones showed good activity as PDE4 inhibitors. The most potent compounds in this series showed an IC₅₀ in the nanomolar range. The ability to inhibit TNF-α release in human PBMCs was determined for two representative compounds, finding values in the sub-micromolar range. SARs studies demonstrated that the best arranged groups around the heterocyclic core are 2-chloro-, 2-methyl- and 3-nitrophenyl at position 2, an ethyl ester at position 4 and a small alkyl group at position 6. Molecular modeling studies performed on a representative compound allowed to define its binding mode to the PDE4B isoform.     

Role of Hematopoietic Cells in the Maintenance of Chronic Human Torquetenovirus Plasma Viremia

Many aspects of the life cycle of torquetenoviruses (TTVs) are essentially unexplored. In particular, it is still a matter of speculation which cell type(s) replicates the viruses and maintains the generally high viral loads found in the blood of infected hosts. In this study, we sequentially measured the TTV loads in the plasma of four TTV-positive leukemia patients who were strongly myelosuppressed and then transplanted with haploidentical hematopoietic stem cells. The findings provide clear quantitative evidence for an extremely important role of hematopoietic cells in the maintenance of TTV viremia.Torquetenoviruses (TTVs) are small naked DNA viruses distinguished by a circular single-stranded DNA genome of only 3.8 kb, classified within the newly established family Anelloviridae (7). TTVs have been found in several animal species but do not appear capable of interspecies transmission. Due to their extensive genetic heterogeneity, human TTVs have been operatively subdivided into 5 genogroups and more than 40 genotypes (4). A remarkable feature of these TTVs is their presence in the plasma of nearly all people, regardless of geographical origin, age, and health status, raising many questions about their life cycle and possible pathological implications (2, 5). Plasma loads of TTVs vary extensively in both healthy and diseased individuals, usually ranging between 10³ and 10⁷ DNA copies per ml of plasma. However, some patients, including those with selected inflammatory or neoplastic disorders, transplant recipients, and human immunodeficiency virus-infected individuals, have a tendency to carry especially high burdens of TTVs (1, 6, 13, 22-24).By studying the dynamics of TTV viremia in individuals treated with alpha interferon for hepatitis C, the kinetics of virus replication was found to be quite high, with numbers of virions released into plasma and cleared from it daily on the same order of magnitude as other chronic plasma viremia-inducing viruses, such as the hepatitis B, hepatitis C, and human immunodeficiency viruses (16). Yet, due to considerable difficulties encountered in propagating TTVs in culture and in distinguishing the virions passively adsorbed onto the cells from the ones replicating inside cells, the tissue or tissues where these large numbers of TTV virions originate have yet to be established. Given that the amino acid compositions of the capsid protein believed to mediate viral adsorption to cells are quite diverse in different TTVs (2, 3, 9), it is also possible that permissive cells vary depending on the TTV considered. Relevant studies are limited. Short-term cultures of phytohemagglutinin-stimulated peripheral lymphocytes, but not resting lymphocytes were found to permit a measurable level of TTV replication (15, 18), indicative of at least a moderate degree of lymphotropism. On the other hand, the detection of replicative forms of TTV DNA in several tissues, including bone marrow, peripheral blood mononuclear cells, and liver, has suggested that TTVs might be polytropic in nature (2, 21).In 1999, Kanda et al. (10), researching TTV plasma of bone marrow transplant recipients with a qualitative PCR, noticed that 5 out of 6 previously positive patients tested negative in a sampling collected during the myelosuppressed period and became positive again after graft reconstitution, leading them to suggest that TTV might replicate mainly in hematopoietic cells. In the present study, we further developed this observation by measuring the TTV load in sequential plasma samples obtained from four TTV-positive leukemia patients undergoing hematopoietic stem cell transplantation. This procedure basically consists of a myeloablative conditioning regimen (chemotherapy plus radiotherapy) followed by reinfusion of a positively selected CD34⁺ stem cell population. The findings are of interest because, in addition to confirming the decrease of TTV load observed by Kanda et al., they shed light on the kinetics of the effect, thus providing a better insight onto the role of hematopoietic cells in the maintenance of TTV viremia and on the life cycle of TTV in general.Table ​Table11 summarizes the main characteristics of the patients selected for the study. They were treated with 10 Gy total-body irradiation (TBI) on day 0 and received 5 mg/kg/day thiotepa on days 2 and 3, 40 mg/m²/day fludarabine on days 3 to 7, and 1.2 mg/kg/day antithymocyte globulin on days 4 to 8, and then, on day 10, they received the indicated numbers of positively selected CD34⁺ hematopoietic stem cells from HLA-haploidentical donors. Peripheral blood samples were collected for TTV studies immediately before TBI and at selected times for the next 30 days, and plasma was stored in aliquots at −80°C until DNA extraction. The assay used for TTV quantification was a previously described highly sensitive TaqMan real-time PCR having the potential to detect and quantitate all hitherto recognized genetic forms of the virus (15, 16). All samples from each patient were assayed in a single run and in triplicate, and at least two independent DNA extractions for each sample were examined. The DNA extracts obtained at time zero were also typed with a previously described panel of five distinct PCR assays (12), each specific for one of the genogroups into which TTVs are subdivided. At the start of the study, the patients had viral loads ranging from 4.7 to 6.8 log copies per ml of plasma and harbored between 1 and 3 TTV genogroups (Table ​(Table1).1). In particular, all carried genogroup 1, which is highly represented in our area (12), and two carried one or two further genogroups. Consistent with previous findings (12), the patient who harbored three genogroups was the one with the highest viral load. As shown by Fig. ​Fig.1,1, in all four patients, TBI was followed by a steady decline of TTV viremia that continued for at least 22 days and progressively brought the virus to levels very close to the detection limit of the detection/quantitation method used, corresponding to values ranging between 0.003 (patient 3) and 0.00009 (patient 1) of the loads present prior to TBI. However, in no instance did the viral loads go below the limit of sensitivity of the assay (2 × 10² TTV DNA copies per ml of plasma). Although the size of the study does not permit firm conclusions on this aspect, it is noteworthy that the extent of decline was unrelated to the type and number of infecting TTV genogroup(s) originally present in the patients.Open in a separate windowFIG. 1.Plasma TTV loads and WBC counts in the peripheral blood of the 4 patients (Pt. 1 to 4) enrolled in the study. The arrow indicates the day the patients were infused with CD34⁺ hematopoietic stem cells from HLA-haploidentical donors. The horizontal broken line represents the lower limit of sensitivity of the TTV detection method used.

TABLE 1.

Relevant parameters of the patients enrolled

Neurohormones and inflammatory mediators in patients with heart failure undergoing cardiac resynchronization therapy: time courses and prediction of response

Despite interest in neurohormonal activation as a determinant of prognosis in chronic heart failure (CHF) and as a target for pharmacological treatments, data are lacking on the time-related effects of electrical cardiac resynchronization therapy (CRT) on a broad spectrum of neurohormones and cytokines. The aim of this study was to assess time-courses and extents of changes within the neurohormonal profile of CHF patients treated with CRT. We performed a prospective follow-up study in 32 patients with NYHA class III-IV CHF to investigate the effects of CRT on a broad panel of neurohormones proposed for characterization of CHF patients. Levels of atrial and brain natriuretic peptides (ANP, BNP), epinephrine, norepinephrine, aldosterone, plasma renin activity, IL-6, TNF, soluble receptors sTNFR1 and 2, and chromogranin A were assessed before implantation and after 3 months of CRT; when feasible, measurements were also performed at 1 week, 1 month and 12 months (clinical evaluation, echocardiography and ECG were also performed at each time-point). The results showed that at 3 months improvement in NYHA class and echographically assessed left ventricular (LV) reverse structural remodeling were accompanied by significant reductions versus baseline in ANP and BNP, but not in other neurohormones. Moreover a baseline ANP concentration < or = 150 pg/ml was a good predictor of response to CRT in terms of NYHA class reduction and reverse LV remodeling. In conclusion 3 months of CRT significantly reduce natriuretic peptides concentrations, while values of other neurohormones and inflammatory cytokines are relatively unvaried. A baseline ANP concentration < or = 150 pg/ml might be a clinically useful predictor of medium-term response to CRT.     

Biofouling of reverse osmosis membranes used in river water purification for drinking purposes: analysis of microbial populations

Biofouling in water treatment processes represents one of the most frequent causes of plant performance decline. Investigation of clogged membranes (reverse osmosis membranes, microfiltration membranes and ultrafiltration membranes) is generally performed on fresh membranes. In the present study, a multidisciplinary autopsy of a reverse osmosis membrane (ROM) was conducted. The membrane, which was used in sulfate-rich river water purification for drinking purposes, had become inoperative after 6 months because of biofouling and was later stored for 18 months in dry conditions before analysis. SSU rRNA gene library construction, clone sequencing, T-RFLP, light microscope, and scanning electron microscope (SEM) observations were used to identify the microorganisms present on the membrane and possibly responsible for biofouling at the time of removal. The microorganisms were mainly represented by bacteria belonging to the phylum Actinobacteria and by a single protozoan species belonging to the Lobosea group. The microbiological analysis was interpreted in the context of the treatment plant operations to hypothesize as to the possible mechanisms used by microorganisms to enter the plant and colonize the ROM surface.     

The antibiotic Furvina? targets the P-site of 30S ribosomal subunits and inhibits translation initiation displaying start codon bias

Furvina®, also denominated G1 (MW 297), is a synthetic nitrovinylfuran [2-bromo-5-(2-bromo-2-nitrovinyl)-furan] antibiotic with a broad antimicrobial spectrum. An ointment (Dermofural®) containing G1 as the only active principle is currently marketed in Cuba and successfully used to treat dermatological infections. Here we describe the molecular target and mechanism of action of G1 in bacteria and demonstrate that in vivo G1 preferentially inhibits protein synthesis over RNA, DNA and cell wall synthesis. Furthermore, we demonstrate that G1 targets the small ribosomal subunit, binds at or near the P-decoding site and inhibits its function interfering with the ribosomal binding of fMet-tRNA during 30S initiation complex (IC) formation ultimately inhibiting translation. Notably, this G1 inhibition displays a bias for the nature (purine vs. pyrimidine) of the 3′-base of the codon, occurring efficiently only when the mRNA directing 30S IC formation and translation contains the canonical AUG initiation triplet or the rarely found AUA triplet, but hardly occurs when the mRNA start codon is either one of the non-canonical triplets AUU or AUC. This codon discrimination by G1 is reminiscent, though of opposite type of that displayed by IF3 in its fidelity function, and remarkably does not occur in the absence of this factor.     

Identification of Single Nucleotide Polymorphisms in the Nicastrese Goat and Sardinia Sheep Mannose-Binding Lectin

This study was undertaken to detect polymorphisms in the goat and sheep mannose-binding lectin encoding gene (MBL2) and to explore allelic variability of this gene in these two species. The analysis and comparison of the sequences obtained from sheep showed 13 polymorphic sites, six in the promoter and seven in exon 1, four of which were of the missense type. In the goats, 12 polymorphic sites were detected, five intronic, five in the promoter, and one exonic. The exon site was responsible for an amino acid change. Mutations detected at the MBL2 locus in the sheep are of particular interest, being potentially responsible for the alterations of gene expression. A population survey involved 102 ewes of the Sardinian breed and 218 goats of the Nicastrese breed, all reared in southern Italy.     

Homers regulate calcium entry and aggregation in human platelets: a role for Homers in the association between STIM1 and Orai1

Homer is a family of cytoplasmic adaptor proteins that play different roles in cell function, including the regulation of G-protein-coupled receptors. These proteins contain an Ena (Enabled)/VASP (vasodilator-stimulated phosphoprotein) homology 1 domain that binds to the PPXXF sequence motif, which is present in different Ca2?-handling proteins such as IP3 (inositol 1,4,5-trisphosphate) receptors and TRPC (transient receptor potential canonical) channels. In the present study we show evidence for a role of Homer proteins in the STIM1 (stromal interaction molecule 1)-Orai1 association, as well as in the TRPC1-IP3RII (type II IP3 receptor) interaction, which might be of relevance in platelet function. Treatment of human platelets with thapsigargin or thrombin results in a Ca2?-independent association of Homer1 with TRPC1 and IP3RII. In addition, thapsigargin and thrombin enhanced the association of Homer1 with STIM1 and Orai1 in a Ca2?-dependent manner. Interference with Homer function by introduction of the synthetic PPKKFR peptide into cells, which emulates the proline-rich sequences of the PPXXF motif, reduced STIM1-Orai1 and TRPC1- IP3RII associations, as compared with the introduction of the inactive PPKKRR peptide. The PPKKFR peptide attenuates thrombin-evoked Ca2? entry and the maintenance of thapsigargin-induced store-operated Ca2? entry. Finally, the PPKKFR peptide attenuated thrombin-induced platelet aggregation. The findings of the present study support an important role for Homer proteins in thrombin-stimulated platelet function, which is likely to be mediated by the support of agonist-induced Ca2? entry.     

Analysis of endothelial protein C receptor gene and metabolic profile in Prader-Willi syndrome and obese subjects

The endothelial protein C receptor (EPCR) has a critical role in the regulation of anticoagulant and anti-inflammatory functions of activated protein C (APC). Abnormalities in EPCR might be associated with an increased risk of thrombosis. In this respect, a 23 bp insertion in the exon 3 of the EPCR gene predicts a truncated protein which cannot bind APC. High levels of C-reactive protein (CRP), a strong predictor of cardiovascular events, are found both in the obese and in subjects with Prader-Willi syndrome (PWS). Several cardiovascular risk factors are already present in prepubertal PWS children, but it is uncertain which mechanism contributes to the increased risk of cardiovascular disease in PWS. We analyzed the distribution of 23 bp insertion in the EPCR gene in 81 overweight and obese PWS subjects, 52 adults and 29 children, and in 58 overweight and obese children and adolescents (controls). We found that 1/58 (1.7%) of the controls was heterozygous for the 23 bp insertion, while this mutation was never found in PWS subjects. Furthermore, we evaluated CRP levels, glucose, insulin, and lipid profile, and we found higher CRP values in PWS adults with respect to children with PWS and controls, and a better insulin sensitivity in all PWS subjects than in the controls. This study suggests that in PWS subjects there is no predisposition to develop thrombotic events in association with EPCR gene alteration and demonstrates substantial differences regarding metabolic and inflammatory profile between PWS and non-PWS obese children, with further impairment in adults with PWS.     

Concomitant chemo-radiotherapy for unresectable oesophageal cancer: A mono-institutional study on 40 patients

Background/AimTo analyse clinical response, overall (OS) and disease free survival (DFS) and toxicity in patients with unresectable oesophageal cancer treated by concomitant chemo-radiotherapy (CRT).Materials and methodsForty patients with stage IIa–IVa biopsy proven oesophageal carcinoma were treated with CRT. All patients were studied with endoscopy and CT and judged unresectable after multidisciplinary discussion. CRT consisted of 3 cycles of cisplatin 100 mg/m² or carboplatin 300 mg/m² on day 1 and 5-fluorouracil 1000 mg/m² as a continuous infusion of 96 h associated with concurrent 3D-conformal RT. By using 15 MeV X-rays, a total dose of 60–66 Gy was delivered with daily fractions of 1.8–2.0 Gy.ResultsComplete response (CR), partial response (PR) and no response (NR) were observed in 50%, 20% and 20% of cases, respectively. Of the 20 patients with CR, 15 developed loco-regional recurrent disease. OS and DFS rates at 3 and 5 years were 38%, 8%, 49% and 10%, respectively. Total radiation dose ≥60 Gy improved loco-regional control and complete response (CR vs. PR + NR; p = 0.004) influenced both DFS and loco-regional control. Grade 3 gastrointestinal and haematological acute toxicity occurred in 3/40 patients (7.5%). One patient developed grade 4 renal failure. Late toxicity was reported in 2/40 patients (5.0%), consisting of grade 3 radiation pneumonitis.ConclusionsConcomitant CRT for unresectable oesophageal cancer can result in an acceptable loco-regional control with limited toxicity. Response after treatment and total radiation dose influenced the outcome.