Modification of translation factor aIF5A from Sulfolobus solfataricus.

Eukaryotic eIF5A and its bacterial orthologue EF-P are translation elongation factors whose task is to rescue ribosomes from stalling during the synthesis of proteins bearing particular sequences such as polyproline stretches. Both proteins are characterized by unique post-translational modifications, hypusination and lysinylation, respectively, which are essential for their function. An orthologue is present in all Archaea but its function is poorly understood. Here, we show that aIF5A of the crenarchaeum Sulfolobus solfataricus is hypusinated and forms a stable complex with deoxyhypusine synthase, the first enzyme of the hypusination pathway. The recombinant enzyme is able to modify its substrate in vitro resulting in deoxyhypusinated aIF5A. Moreover, with the aim to identify the enzyme involved in the second modification step, i.e. hypusination, a set of proteins interacting with aIF5A was identified.

Action against vaccine-preventable infectious diseases and tuberculosis in Nomad Camps: the experience of a Local Health Unit in Rome.

In Italy the highest incidence of Tuberculosis (TB) cases is in young adult migrants. In 2011, the sanitarystaff of the Local Health Unit (ASL) Roma A promoted a vaccination campaign conducting several public health interventions in Nomad Camps. After notification of a case of TB in the Camp of Via Salaria, out of 357 Mantoux skin tests performed, 93 were positive (26%); subsequently, 5 subjects with radiographic positivity were hospitalized. The vaccination campaign was carried out to prevent the spread of infectious diseases in immigrant communities at high risk of contagion and to avoid the consequent transmission in the host country. As a result, vaccinations coverage among the residents of the Camps increased: 367 vaccinated subjects (30% more than previous year) and 612 administered vaccinations.

Mechanistic understanding of gene delivery mediated by highly efficient multicomponent envelope-type nanoparticle systems.

We packaged condensed DNA/protamine particles in multicomponent envelope-type nanoparticle systems (MENS) combining different molar fractions of the cationic lipids 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and 3ß-[N-(N,N-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) and the zwitterionic lipids dioleoylphosphocholine (DOPC) and dioleoylphosphatidylethanolamine (DOPE). Dynamic light scattering (DLS) and microelectrophoresis allowed us to identify the cationic lipid/DNA charge ratio at which MENS are small sized and positively charged, while synchrotron small-angle X-ray scattering (SAXS) and atomic force microscopy (AFM) revealed that MENS are well-shaped DNA/protamine particles covered by a lipid monobilayer. Transfection efficiency (TE) experiments indicate that a nanoparticle formulation, termed MENS-3, was not cytotoxic and highly efficient to transfect Chinese hamster ovary (CHO) cells. To rationalize TE, we performed a quantitative investigation of cell uptake, intracellular trafficking, endosomal escape, and final fate by laser scanning confocal microscopy (LSCM). We found that fluid-phase macropinocytosis is the only endocytosis pathway used by MENS-3. Once taken up by the cell, complexes that are actively transported by microtubules frequently fuse with lysosomes, while purely diffusing systems do not. Indeed, spatiotemporal image correlation spectroscopy (STICS) clarified that MENS-3 mostly exploit diffusion to move in the cytosol of CHO cells, thus explaining the high TE levels observed. Also, MENS-3 exhibited a marked endosomal rupture ability resulting in extraordinary DNA release. The lipid-dependent and structure-dependent TE boost suggests that efficient transfection requires both the membrane-fusogenic activity of the nanocarrier envelope and the employment of lipid species with intrinsic endosomal rupture ability.

Cadmium, lead, and chromium in large game: a local-scale exposure assessment for hunters consuming meat and liver of wild boar.

Heavy metals are ubiquitous in soil, water, and air. Their entrance into the food chain is an important environmental issue that entails risks to humans. Several reports indicate that game meat can be an important source of heavy metals, particularly because of the increasing consumption of game meat, mainly by hunters. We performed an exposure assessment of hunters and members of their households, both adults and children, who consumed wild boar (WB) meat and offal. We estimated the amount of cadmium, lead, and chromium in the tissues of WB hunted in six areas within Viterbo Province (Italy) and gathered data on WB meat and offal consumption by conducting specific diet surveys in the same areas. The exposure to cadmium, lead, and chromium was simulated with specifically developed Monte Carlo simulation models. Cadmium and lead levels in WB liver and meat harvested in Viterbo Province (Italy) were similar to or lower than the values reported in other studies. However, some samples contained these metals at levels greater then the EU limits set for domestic animals. The chromium content of meat or liver cannot be evaluated against any regulatory limit, but our results suggest that the amounts of this metal found in WB products may reflect a moderate environmental load. Our survey of the hunter population confirmed that their consumption of WB meat and liver was greater than that of the general Italian population. This level of consumption was comparable with other European studies. Consumption of WB products contributes significantly to cadmium and lead exposure of both adults and children. More specifically, consumption of the WB liver contributed significantly to total cadmium and lead exposure of members of the households of WB hunters. As a general rule, liver consumption should be kept to a minimum, especially for children living in these hunter households. The exposure to chromium estimated for this population of hunters may be considered to be safe. However, a specific and complete assessment of chromium speciation in relevant dietary and environmental situations should be conducted.

CCL16 enhances the CD8+ and CD4+ T cell reactivity to human HER-2 elicited by dendritic cells loaded with rat ortholog HER-2.

T cells from HLA-A2+ healthy donors were co-cultured with autologous dendritic cells (DC) loaded with apoptotic tumor cells expressing rat neu, and were induced to mature by tumor necrosis factor (TNF)alpha and interleukin (IL)-1beta (mDC(neu)) or by the CCL16 chemokine (CCL16/mDC(neu)). Priming by CCL16/mDC(neu) induces a larger population of T cells that express cytoplasmatic interferon (IFN)gamma, TNFalpha, perforin and granzyme B compared to those primed by mDC(neu). T cells primed by CCL16/mDC(neu) release IFNgamma in response to human HER-2+ cells and kill human HER-2+ target cells more efficiently than those primed by mDC(neu). Our results show that both the loading of DC with xenogeneic rat neu and their maturation by CCL16 are two issues of critical importance for the elicitation of an effective response to human HER-2 in T cells from normal donors.

The Marigliano-Cacciafesta polypathology scale (MCPS): a tool for predicting the risk of developing disability.

The purpose of the work was to verify whether our MCPS can be a tool for predicting the risk of developing disabilities. We considered 45 elderly subjects divided into three groups of 15 subjects each. Group 1 consisted of cases with a "moderate-severe" degree of polypathology, with no associated condition of disability evaluated by means of the activities of daily living (ADL). Group 2 contained cases with a "moderate" degree of polypathology (with no associated condition of ADL disability). The Group 3 was the control group with a "mild" degree of polypathology (with no disability associated with ADL). All subjects were re-evaluated after 6 and 12 months. Both Groups 1 and 2 of cases over time developed greater disabilities, compared to the control Group 3; in particular, the subjects with "moderate-severe" polypathology were more disabled after 12 months.

The Marigliano-Cacciafesta polypathological scale: a tool for assessing fragility.

The aim of our studies was to establish a standard method of assessment that allows an early identification of frailty in the elderly, i.e., to predict who are at risk of developing disabilities, in order to be able to intervene with preventive global and individualized measures. A new multidimensional scale called Marigliano-Cacciafesta polypathological scale (MCPS) was used on 180 elderly people, together with the Barthel index (BI), the global evaluation functional index (GEFI), the geriatric depression scale (GDS), the mini mental state examination (MMSE), the mini nutritional assessment (MNA), and the Tinetti test. A strongly significant statistical correlation was found between the MCPS and the nutritional state, mood level, motor functionality, level of disability and global functionality. As the fragile patients are at a risk to develop disabilities, we think that our scale can be a significant contribution to the multidimensional geriatric assessment (MGA), aimed at identifying and quantifying the parameter of fragility of each patient, an information which should be known, if we intend to introduce preventive measures.

Combination of Coenzyme Q10 Intake and Moderate Physical Activity Counteracts Mitochondrial Dysfunctions in a SAMP8 Mouse Model.

Aging skeletal muscles are characterized by a progressive decline in muscle mass and muscular strength. Such muscular dysfunctions are usually associated with structural and functional alterations of skeletal muscle mitochondria. The senescence-accelerated mouse-prone 8 (SAMP8) model, characterized by premature aging and high degree of oxidative stress, was used to investigate whether a combined intervention with mild physical exercise and ubiquinol supplementation was able to improve mitochondrial function and preserve skeletal muscle health during aging. 5-month-old SAMP8 mice, in a presarcopenia phase, have been randomly divided into 4 groups (n = 10): untreated controls and mice treated for two months with either physical exercise (0.5 km/h, on a 5% inclination, for 30 min, 5/7 days per week), ubiquinol 10 (500 mg/kg/day), or a combination of exercise and ubiquinol. Two months of physical exercise significantly increased mitochondrial damage in the muscles of exercised mice when compared to controls. On the contrary, ubiquinol and physical exercise combination significantly improved the overall status of the skeletal muscle, preserving mitochondrial ultrastructure and limiting mitochondrial depolarization induced by physical exercise alone. Accordingly, combination treatment while promoting mitochondrial biogenesis lowered autophagy and caspase 3-dependent apoptosis. In conclusion, the present study shows that ubiquinol supplementation counteracts the deleterious effects of physical exercise-derived ROS improving mitochondrial functionality in an oxidative stress model, such as SAMP8 in the presarcopenia phase.

Second allogeneic bone marrow transplantation in acute leukemia: a multicenter study from the Gruppo Italiano Trapianto Di Midollo Osseo (GITMO).

Thirty-eight second allogeneic bone marrow transplants (BMT) for acute leukemia relapsed after first BMT were performed in 13 Italian centers between 1987 and 1994. Twenty-one patients had acute myelogenous leukemia (AML), 17 acute lymphoblastic leukemia (ALL); at second BMT 24 patients were in complete remission (CR) and 14 in relapse. The median time to relapse after first BMT was 10 months (range 1-70). Grade II or greater acute graft-versus-host disease (GVHD) after second transplant occurred in 34.2% of patients and a chronic GVHD in 31.5% of patients. Twenty-four patients died: seven from early transplant-related mortality (TRM), 13 from relapse and four from late toxicity. As of 31 July 1996, at a median follow-up of 47 months (range 22-85), there are 14 survivors. The three-year probability of TRM, relapse and event-free survival (EFS) is 28%, 40% and 42% respectively. In 20 of 27 evaluable patients, remission duration after second BMT was longer than after the first BMT. A diagnosis of AML was correlated with a better outcome. These data support the usefulness of second allograft in selected patients with AML relapsing after a first BMT.

Inhibition of mammary carcinogenesis by systemic interleukin 12 or p185neu DNA vaccination in Her-2/neu transgenic BALB/c mice.

Because BALB/c mice transgenic for the rat Her-2/neu oncogene develop multifocal carcinomas in all mammary glands by week 33, they constitute an aggressive model for investigation of treatments designed to oppose mammary carcinogenesis. Nonspecific immune reaction elicited by systemic interleukin (IL)-12 both delayed the appearance of the first tumor and reduced the number of glands affected. However, only 5% of mice were tumor free at week 33. On the other hand, specific vaccination with plasmids encoding for the rat p185neu resulted in a further delay, so much so that 58% of mice were tumor free at week 33. No CTL response was evoked in either IL-12-treated or DNA-vaccinated mice, whereas an anti-rat p185neu antibody response was evident in the latter. Pathological examinations showed that in both IL-12-treated and DNA-vaccinated mice, the tumor growth area was infiltrated by reactive cells associated with expression of endothelial adhesion molecules and antiangiogenic proinflammatory cytokines. In the vaccinated mice, reduction of the number of cells expressing rat p185neu was combined with down-regulation of its membrane expression and even a marked inhibition in development of the terminal ductal lobular units. The reactive infiltrate in vaccinated mice contained numerous granulocytes that likely played an antiangiogenic and angiodestructive role and also joined other cells in the antibody-mediated killing of the r-p185neu+ cells. These results suggest that the elicitation of nonspecific and specific immunity could be beneficially used in individuals with a high risk of developing tumors.

Pyrimidine nucleotidases from human erythrocyte possess phosphotransferase activities specific for pyrimidine nucleotides.

Two cytoplasmic forms of pyrimidine nucleotidase (PN-I and PN-II) have been purified from human erythrocytes to apparent homogeneity and partially characterized. They preferentially hydrolyse pyrimidine 5'-monophosphates and 3'-monophosphates respectively. PN-I and PN-II operate as interconverting activities, capable of transferring the phosphate from the pyrimidine nucleoside monophosphate donor(s) to various nucleoside acceptors, including important drugs like 3'-azido-3'-deoxy-thymidine (AZT), cytosine-beta-D-arabinofuranoside (AraC) and 5-fluoro-2'-deoxy-uridine (5FdUrd), pyrimidine analogues widely used in chemotherapy. Kinetic analysis showed linear behaviour for both PN-I and PN-II. PN-I phosphotransferase activity revealed higher affinity for oxynucleosides with respect to deoxy-nucleosides, whereas the contrary seems to be true for PN-II. These results show for the first time that soluble pyrimidine nucleotidases are endowed with pyrimidine-specific phosphotransferase activity.

Pyridine dinucleotide biosynthesis in archaebacteria: presence of NMN adenylyltransferase in Sulfolobus solfataricus.

The enzyme NMN adenylyltransferase, leading to NAD synthesis, has been observed for the first time in soluble extracts from the extreme acidothermophilic archaeon Sulfolobus solfataricus. Comparison of its molecular and kinetic properties with those of the enzyme isolated from prokaryotes and eukaryotes revealed significant differences, knowledge of which may contribute to the understanding of metabolic evolutionary mechanisms. The thermophilic enzyme shows a molecular mass of about 66,000 and an isoelectric point of 5.4. The Km values for ATP, NMN and nicotinic acid mononucleotide are 0.08 microM, 1.4 microM and 17 microM, respectively. The enzyme shows a remarkable degree of thermophilicity, with an activation energy of 95 kJ/mol.

Identification and characterization of YLR328W, the Saccharomyces cerevisiae structural gene encoding NMN adenylyltransferase. Expression and characterization of the recombinant enzyme.

The enzyme nicotinamide mononucleotide (NMN) adenylyltransferase (EC 2.7.7.1) catalyzes the transfer of the adenylyl moiety of ATP to NMN to form NAD. A new purification procedure for NMN adenylyltransferase from Saccharomyces cerevisiae provided sufficient amounts of enzyme for tryptic fragmentation. Through data-base search a full matching was found between the sequence of tryptic fragments and the sequence of a hypothetical protein encoded by the S. cerevisiae YLR328W open reading frame (GenBank accession number U20618). The YLR328W gene was isolated, cloned into a T7-based vector and successfully expressed in Escherichia coli BL21 cells, yielding a high level of NMN adenylyltransferase activity. The purification of recombinant protein, by a two-step chromatographic procedure, resulted in a single polypeptide of 48 kDa under SDS-PAGE, in agreement with the molecular mass of the hypothetical protein encoded by YLR328W ORF. The N-terminal sequence of the purified recombinant NMN adenylyltransferase exactly corresponds to the predicted sequence. Molecular and kinetic properties of recombinant NMN adenylyltransferase are reported and compared with those already known for the enzyme obtained from different sources.

Synechocystis sp. slr0787 protein is a novel bifunctional enzyme endowed with both nicotinamide mononucleotide adenylyltransferase and 'Nudix' hydrolase activities.

Synechocystis sp. slr0787 open reading frame encodes a 339 residue polypeptide with a predicted molecular mass of 38.5 kDa. Its deduced amino acid sequence shows extensive homology with known separate sequences of proteins from the thermophilic archaeon Methanococcus jannaschii. The N-terminal domain is highly homologous to the archaeal NMN adenylyltransferase, which catalyzes NAD synthesis from NMN and ATP. The C-terminal domain shares homology with the archaeal ADP-ribose pyrophosphatase, a member of the 'Nudix' hydrolase family. The slr0787 gene has been cloned into a T7-based vector for expression in Escherichia coli cells. The recombinant protein has been purified to homogeneity and demonstrated to possess both NMN adenylyltransferase and ADP-ribose pyrophosphatase activities. Both activities have been characterized and compared to their archaeal counterparts.

Structure and function of nicotinamide mononucleotide adenylyltransferase.

The enzyme nicotinamide mononucleotide adenylyltransferase (NMNAT), a member of the nucleotidyltransferase alpha/beta phosphodiesterase superfamily, catalyzes the reaction NMN + ATP = NAD + PPi, representing the final step in the biosynthesis of NAD, a molecule playing a fundamental role as a cofactor in cellular redox reactions. NAD also serves as the substrate for reactions involved in important regulatory roles, such as protein covalent modifications, like ADP-ribosylation reactions, as well as Sir2 histone deacetylase, a recently discovered class of enzymes involved in the regulation of gene silencing. This overview describes the most recent findings on NMNATs from bacteria, archaea, yeast, animal and human sources, with detailed consideration of their major kinetic, molecular and structural features. On this regard, the different characteristics exhibited by the enzyme from the various species are highlighted. The possibility that NMNAT may represent an interesting candidate as a target for the rational design of selective chemotherapeutic agents has been suggested.

Outcome after cessation of therapy in childhood acute lymphoblastic leukaemia. The Associazione Italiana Ematologia ed Oncologia Pediatrica (AIEOP).

A total of 2192 children with acute lymphoblastic leukaemia who had reached cessation of therapy in complete remission were followed for a median time of 52 months after treatment suspension. Of the 485 relapses observed, 62.3% occurred in the first year off therapy and 68.9% involved the bone marrow. Eight relapses were reported more than 5 years (62-143 months) after treatment withdrawal. Males fared worse than females consistently, experiencing 1.5 times more relapses (P < 0.0001). Thirteen patients died in continuous complete remission, 5 because of non-neoplastic central nervous system complications. There were 11 second solid malignancies, 8 of them in the central nervous system; 9 subjects presented an haematopoietic malignancy after ALL. The projected event-free survival at 8 years is 73%. Twenty-two of the 171 young adults (age > 20 years) were married and 16 have had 21 healthy children. Twenty-four per cent of patients experienced an unfavourable event. Relapses accounted for 93% of failures. Central nervous system late effects and second malignancies were the major causes of non-leukaemic morbidity and mortality.

Binding of feline immunodeficiency viral gag-p24 polypeptide to nonimmune Igs.

Available data on the existence of lentivirus proteins with properties of unconventional Ag for B cells, have been so far restricted to human immunodeficiency virus (i.e. gp-120 of HIV-I). By using biotinylated-MAbs-anti-biotin IgG as readout system, we now report that gag-p24 antigen, either assembled in feline immunodeficiency virus (FIV) particles or expressed as recombinant polypeptide (rec.p24) may bind to nonimmune IgGs purified from mouse or cat sera. Moreover, FACS scanning experiments are consistent with the possibility that rec.p24 interacts with surface-Ig in a sub-population (5-6%) of rodent B cells. We hypothesize that gag-p24 peptide encoded regions may bind to unconventional Ig sites or, alternatively, that they may represent 'public' epitopes for natural polyreactive antibody.

The antitumor drug, 1,3-bis(2-chloroethyl)-1-nitroso-urea, inactivates human nicotinamide mononucleotide adenylyltransferase.

Nicotinamide mononucleotide (NMN) adenylyltransferase (EC 2.7.7.1) from human placenta is rapidly inactivated by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). A similar inactivation is observed with other C- and N-nitroso compounds. The inactivation by BCNU is dependent on incubation time, temperature and BCNU concentration. Protective reagents for -SH groups, dithiothreitol and beta-mercaptoethanol, and the substrate NMN are very effective in protecting NMN adenylyltransferase from BCNU inactivation and in preserving its catalytic properties, while ATP is less efficient. Incubation of BCNU-inactivated and dialysed NMN adenylyltransferase with dithiothreitol results in a partial recovery of the enzymatic activity.

Theoretical model for the superconducting and magnetically ordered borocarbides

We present a theory of superconductivity in the presence of a general magnetic structure in a form suitable for the description of complex magnetic phases encountered in borocarbides. The theory, complemented with some details of the band structure and with the magnetic phase diagram, may explain the nearly reentrant behavior and the anisotropy of the upper critical field of HoNi2B2C. The onset of the helical magnetic order depresses superconductivity via the reduction of the interaction between phonons and electrons caused by the formation of magnetic Bloch states. At mean field level, no additional suppression of superconductivity is introduced by the incommensurability of the helical phase.

Mismatched T cell-depleted hematopoietic stem cell transplantation for children with high-risk acute leukemia.

The aim of this study was to extend allogeneic hematopoietic stem cell transplantation to leukemia patients without a matched donor. To prevent graft failure, large doses of T cell-depleted hematopoietic stem cells were transplanted following a highly myeloablative and immunosuppressive conditioning regimen. Fifteen children with high-risk acute leukemia received T cell-depleted hematopoietic stem cells from full-haplotype mismatched family members after a conditioning regimen that included single-dose TBI, thiotepa, ATG and fludarabine. To prevent GVHD, marrow cells were T-depleted by soybean agglutinin and E-rosetting, peripheral blood cells by E-rosetting followed by positive selection of the CD34+ cells. No post-transplant prophylaxis for GVHD was administered. In all patients full donor-type engraftment was achieved. None of the evaluable patients developed either acute or chronic GVHD. Regimen-related toxicity was minimal. Five patients are alive and event-free at a median follow-up of 18 months (range 13-28). All surviving patients have a good quality of life. Seven patients have relapsed. This study shows that GVHD and graft failure, which limited the use of full-haplotype mismatched bone marrow transplants, have been overcome. Since almost all children have a mismatched relative, advances in this area should make mismatched transplants a routine consideration for patients with high-risk leukemia without a matched related or unrelated donor.