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HIV-1 Rev mediates the nuclear export of intron-containing viral RNA transcripts and is essential for viral replication. Rev is imported into the nucleus by the host protein Importin β (Impβ), but how Rev associates with Impβ is poorly understood. Here we report biochemical, biophysical and structural studies of the Impβ/Rev complex. Gel shift, native mass spectrometry and isothermal titration calorimetry data reveal that Impβ binds two Rev monomers through independent binding sites. Small-angle X-ray scattering (SAXS) data suggest that the HEAT repeats of Impβ retain an extended conformation upon binding Rev, which according to NMR data is primarily recognized through its helical hairpin domain. Peptide scanning data and charge-reversal mutations identify the N-terminal tip of Rev helix α2 within Rev’s Arginine-Rich Motif (ARM) as a primary Impβ binding epitope. Crosslinking mass spectrometry and compensatory mutagenesis data combined with molecular docking simulations suggest a structural model in which one Rev monomer binds to the C-terminal half of Impβ with Rev helix α2 roughly parallel to the HEAT-repeat superhelical axis while the other monomer binds to the N-terminal half. These findings shed light on the molecular basis of Rev recognition by Impβ and highlight an atypical binding behaviour that distinguishes Rev from canonical cellular Impβ cargos.

SUMMARYThe zoonotic risk ofBrucella canishas been considered fairly high for persons who handle breeding dogs in kennels or are exposed to infected animals. Transmission to humans in other circumstances has been thought to be rare. We describe an uncommon outbreak of brucellosis caused byB. caniswhich, to the best of our knowledge, is the first reported in the literature. This outbreak involved six persons (three children and three adults), a bitch and three puppies which had close daily contact with the family. The clinical symptoms of the index case led to an erroneous diagnosis and the infection would have gone undiagnosed if culture had not been positive. This report aims to increase awareness of medical personnel of the need to order screening tests for children, immunodeficient persons or pregnant women presenting with fever of unknown origin, unexplained spleen or liver enlargement or other systemic signs. The emerging zoonotic potential of this disease in urban areas and the need to coordinate canine brucellosis surveillance systems should be evaluated.

This paper presents a thermomicrostructural model for the simulation of the solidification process of an eutectic ductile cast iron. The thermal balance is written at a macroscopic level and takes into account both the structural component being cast and its mould. Models of nucleation and growth represent the evolution of the microstructure, and the microsegregation of silicon is also considered. The resulting formulation is solved using a finite element discretisation of the macrodomain, in which the evolution of the microstructure is taken into account at the Gauss integration points. The numerical results are presented in terms of cooling curves and are compared with available experimental values. Furthermore, the sensitivity of the response with respect to changes in the cooling rate and nucleation parameters are investigated. The agreement between experimental and computational values is acceptable in both quantitative and qualitative aspects. Ways to improve the computational model are sugg...

International audience; Staphylococcus aureus possesses two canonical ABC-importers dedicated to nickel acquisition: the NikABCDE and the CntABCDF systems, active under different growth conditions. This study reports on the extracytoplasmic nickel-binding components SaNikA and SaCntA. We showed by protein crystallography that SaNikA is able to bind either a Ni-(l-His)2 complex or a Ni-(l-His) (2-methyl-thiazolidine dicarboxylate) complex, depending on their availability in culture supernatants. Native mass spectrometry experiments on SaCntA revealed that it binds the Ni(ii) ion via a different histidine-dependent chelator but it cannot bind Ni-(l-His)2. In vitro experiments are consistent with in vivo nickel content measurements that showed that l-histidine has a high positive impact on nickel import via the Cnt system. These results suggest that although both systems may require free histidine, they use different strategies to import nickel.

The present research focuses on the study of the precipitation of free ferrite in ductile iron during cooling through the intercritical temperature range. The main objectives are to recognize the morphology of the free ferrite precipitated in pearlitic ductile iron alloyed with Cu and Mn, and to evaluate the mechanical properties resulting from different microstructures. The microstructures investigated were generated by normalising heat treatments from regular and low austenitising temperatures. The results indicate that different free ferrite morphologies are produced depending on the thermal cycles carried out. As-cast samples present bull's eye morphology, while austenite grain boundary precipitation dominates in recrystallised structures. Low austenitising temperature normalising causes, in the alloys studied, the formation of a fine network of free ferrite precipitated within the pearlitic matrix at the recrystallised austenite grain boundaries. Low austenitising temperature normalising markedly imp...

Summary Von Willebrand disease (VWD) is an inherited bleeding disorder caused by the quantitative or qualitative deficiency of von Willebrand factor (VWF). Replacement therapy with plasma-derived VWF/factor VIII (FVIII) concentrates is required in patients unresponsive to desmopressin. To assess the efficacy, safety and ease of use of a new, volume-reduced (VR) formulation of VWF/FVIII concentrate Haemate® P in patients requiring treatment for bleeding or prophylaxis for recurrent bleeding or for invasive procedures. Pharmacoeconomic variables were also recorded. Data were analysed using descriptive statistics. This was a multicentre, prospective, observational study. Consecutively enrolled patients received Haemate® P VR according to their needs, and were followed for 24 months. Of the 121 patients enrolled, 25.6% had type 3 VWD and more than 40% had severe disease. All patients were followed for 2 years, for a total of 521 visits. On-demand treatment was given to 61.9% of patients, secondary long-term prophylaxis to 25.6% and prophylaxis for surgery, dental or invasive procedures to 45.5%. The response to treatment was rated as good to excellent in >93–99% of interventions. The new formulation was well tolerated by all patients with no report of drug-related adverse events. The switch to volume-reduced Haemate® P was easy to perform and infusion duration was decreased twofold compared with the previous formulation. Volume-reduced Haemate® P was at least as effective and well-tolerated as the previous formulation.

Major questions are whether mothers infected with the Human Immunodeficiency Virus type 1 (HIV-1) transmit the virus through breast milk and the magnitude of the additional transmission risk. The demonstration of a dose-response effect is an epidemiological method to demonstrate causality. Thus, a study was carried out by the Italian Register for HIV Infection in Children on 961 children of known infection status. Duration of breast-feeding was considered as the level of exposure in 168 ever breast-fed children. Results showed that duration of practice significantly increased the risk of transmission. The adjusted infection odds ratio for one day of breast- versus exclusive formula-feeding was 1.19 with narrow confidence limits (1.10-1.28). In a second study by the Register on 556 children of known infection status and derived prospectively, an infection odds ratio of 2.55 (confidence interval: 1.03-6.37) was calculated in breast- versus exclusively formula-fed children. Several lines of evidence, including the above-mentioned data from the Italian Register for HIV Infection in Children, showed a contribution of breast-feeding to mother-to-child HIV-1 transmission. Thus, this practice is now discouraged in HIV-1 infected mothers living in industrialized societies where formula feeding is practical and attainable. Mode of feeding was known in 2183 children enrolled in the Register and born to HIV-1 infected mothers since 1981. It could be observed that feeding habits of at-risk infants changed in Italy in the middle 1980s, when a large majority of subjects was identified at birth.(ABSTRACT TRUNCATED AT 250 WORDS)

Integrin signalling co-ordinates with signalling originating from growth factor receptors in the co-operative control of cell proliferation, survival and migration. Increasing evidence suggests that integrins form physical complexes at the cell membrane with growth factor receptors, giving rise to signalling platforms at the adhesive sites. It is probable that at these sites integrins regulate adhesion and at the same time physically constrain and direct the response to soluble growth factors towards proliferation or survival stimuli. These co-operative effects might depend on integrin ability to activate growth factor receptors. In the present paper, we summarize our recent study showing that integrin-dependent adhesion triggers ligand-independent EGFR (epidermal growth factor receptor) activation to transduce downstream signalling. In addition, we also show that integrin-induced signalling pathways are necessary for EGF-dependent transcriptional response, demonstrating the requirement of the co-operation between cell–matrix adhesion and EGFR to achieve full biological responses.

The localization of HIV-1 proviruses in compositional DNA fractions from 27 AIDS patients during the chronic phase of the disease with depletion of CD4+ and different levels of viremia showed the following. (1) At low viremia, proviruses are predominantly localized in the GC-richest isochores, which are characterized by an open chromatin structure; this result mimics findings on HIV-1 integration in early infected cells in culture. (2) At higher viremia, an increased distribution of proviruses in GC-poor isochores (which match the GC poorness of HIV-1) was found; this suggests a selection of cells in which the ‘isopycnic’ localization leads to a higher expression of proviruses and, in turn, to higher viremia. (3) At the highest viremia, integrations in GC-rich isochores are often predominant again, but generally not at the same level as in (1); this may be the consequence of new integrations from the extremely abundant RNA copies.

Many patterns of mutations selected by HIV-1 protease inhibitors have been described, but in most cases isolates with these patterns have been obtained from pre-clinical studies or after failures of monotherapies. We compared genotype and phenotype in HIV-1 infected patients who have failed more than one PI-including regimen. Phenotypic resistance could arise also in the absence of specific primary mutations and in the presence of different substitutions among those known to confer resistance to ritonavir, indinavir or nelfinavir. The number of secondary mutations was significantly associated with phenotypic resistance for each protease inhibitor. Thus, more study of mutational patterns in heavily pretreated patients is warranted; in the mean time treatment choices might be optimized if phenotyping could integrate genotyping within this setting.

We examined samples from 98 naïve HIV-1 positive patients with the seroconversion period between 1984 and 1997, 64 of whom with a diagnosed primary infection. We observed a progressive increase in the percentage of patients harboring mutations associated with zidovudine resistance, starting from 8% during the period 1987-1994 to 20% and 36% in patients with HIV infection diagnosed during 1996 and 1997 respectively. The small number of patients analyzed in groups 3 and 4 is an important limitation to establish the real increase in the prevalence of mutations we observed during the past two years. However, it is important to underline the trend and, in our opinion, further studies to better define the relevance of such phenomenon in the clinical practice must be performed.

In spite of repeated exposures to HIV, some individuals remain seronegative and apparently uninfected. A variety of mechanisms potentially able to confer resistance to HIV infection, including cell-mediated and (unconventional) humoral immune responses, as well as mutations affecting receptors for virus entry have been considered and analysed. In this article, we want to discuss recent reports on specific immune responses and genetic factors potentially involved in mechanisms of protection, and to present some of our data relative to a cohort of people sexually exposed to HIV-1, but persistently seronegative. These EU (exposed uninfected) individuals can be distinguished from "normal" unexposed controls on the basis of significantly increased frequencies of a number of immunological parameters that might be considered "unconventional" correlates of HIV infection/protection. However, EU individuals are highly heterogeneous since the various unconventional immune responses considered can be present in all possible combinations. Aim of future research will be to ascertain the role of such immune responses in the maintenance of the protection state, or their secondary nature as signals of a particular kind of infection.

The location of HTLV-I (human T-cell leukemia virus type 1) pro viral sequences in the genome of infected human cells was explored by hybridization of a viral probe with compositional fractions of host-cell DNAs. In the twelve cases examined, HTLV-I sequences were absent from the GC-poorest 40% of the host genome (namely, from isochores that are below 39% GC). Transcriptionally inactive proviral sequences were localized in GC-poor isochores (comprised between 39% and 42–44% GC) of the human genome, which are characterized by a constant and low gene concentration. In contrast, transcriptionally active proviral sequences were found in the GC-rich and very GC-rich isochores, which are gene rich, transcriptionally and recombinationally active, and endowed with an open chromatin structure. Since GC-rich isochores are present in R'-bands and very GC-rich isochores form T-bands, these results also provide information on the location of HTLV-I proviral sequences in human chromosomes. The results obtained with HTLV-I are in agreement with the non-random, compartmentalized integration of animal retroviral sequences that had been previously observed in other viral-host systems. They provide, however, much more detailed information on the regional location of proviral sequences in the host genome and on the correlation between their transcription and their location.

It was recently demonstrated that vascular endothelium produces many substances which modulate the vascular tone by acting locally (endothelium derived relaxing factor, angiotensin II, endothelin, prostacyclin). The authors review the physiological regulatory role of these substances and their involvement in cardiovascular diseases. They also consider the interaction between the endothelium derived factors and some cardiovascular drugs (such as calcium antagonists and ACE-inhibitors) and also the mechanisms of the possible protective action of these drugs on the vascular wall.

Essential thrombocythemia (ET) is a chronic myeloproliferative disease, rarely observed in pediatric age, characterized by a persistently increased platelet count. Abnormalities of platelet function observed in ET patients may be, at least in part, responsible for the thrombohemorrhagic complications. The authors report about a pediatric patient affected by ET, showing an abnormal platelet response following stimulation by anti-platelet monoclonal antibody. Such finding may be attributable to a structural abnormality of the platelet fibrinogen receptor or to post-receptor alterations.

The oncoretrovirus HTLV-I is the etiological agent of adult T cell leukemia (ATL) and tropical spastic paraparesis/HLTV-I associated myelopathy (TSP/HAM). In contrast to the human lentiretroviruses, HIV-I and HIV-2, the causative agents of AIDS, HTLV-I is genetically very stable. We report here the molecular characterization of the envelope gene of an HTLV-I variant present in a TSP/HAM patient from Zaïre, raising the question of its relevance to disease or the ethnic and/or geographical origin of the patient.

EVALUATION OF INTRAVENOUS AND INTRAMUSCULAR EFFICACY IN CHILDHOOD IMMUNE THROMBOCYTOPENIC PURPURA

In this study, we used DNA polymorphisms for carrier detection and prenatal diagnosis of hemophilia A in a large group of Italian families. The restriction fragment length polymorphisms (RFLPs) investigated were the intragenic polymorphic Bc/I site within the factor VIII gene; the extragenic multiallelic Taq I system at the St14 locus; and the extragenic Bg/II site at the DX13 locus. The factor VIII probe was informative in 30%, St14 in 82%, and DX13 in 60% of obligate carriers. The combination of factor VIII-Bc/I and St14-Taq I showed that 91% of obligate carriers were heterozygotes for one or both; with all three probes, only 4% of obligate carriers were noninformative. In families clearly segregating for hemophilia A, RFLP analysis allowed us to define the carrier status for the hemophilia A gene in all 27 women tested. RFLP analysis allowed us to exclude the carrier status in 39 of 45 female relatives of sporadic patients. The combination of RFLP analysis and biological assay of factor VIII allowed us to identify a de novo mutation in the maternal grandfather in 7 of 12 of the families with sporadic cases, for which members of three generations were available for study. Nine of 10 couples requesting prenatal diagnosis provided informative RFLP DNA pattern. Carrier status was excluded in two women, two fetuses were shown to be female, and prenatal diagnosis was carried out in five pregnancies by DNA analysis. Prenatal testing was successful in three instances and failed in two because a sufficient amount of chorionic villous DNA was not obtained for the analysis.

It has been known since 1911 (Henze) that the blood of Phallusia mamillata Cuv. contains vanadium, which is found in special cells, called vanadocytes by Webb (1939). Webb estimated that the vanadocytes constitute about 60% of the blood cells and the blood cell volume amounts to between 1 and 2% of the total blood volume. It was found by Henze that the inner medium of the vanadocytes is very acid, due to free sulphuric acid which is present in a concentration of about 3%. According to Webb, the vanadium content of the animal amounts to about 0-15% of the dry weight of the body, and three-quarters of the total amount of vanadium is present in the blood, 15 % in the gut and associated viscera and 10% in the mantle and branchial sac.

(1947). La Courbe De Dissociation De L'oxygene De L'hemoglobine Dans Les Solutions Diluees De Sang. Archives Internationales de Physiologie: Vol. 55, No. 2, pp. 153-162.

The aim of this study is the behavior of Normotest (NT) values in newborns in the first 4 days of life. The study has been carried out between January, 1982, and December, 1984, at the Department of Child Health and Neonatal Medicine - School of Medicine - University of Genoa. The number of infants tested was 1320. 694 were males and 626 females, 529 preterm (G.A. less than 37 weeks) and 791 full term babies. Infants have been tested from one to five times in the first four days of life, with the first evaluation within 12 hours of life. 1215 newborns (92%) presented NT values greater than 20%, 105 babies (8%) had NT below 20% in at least one evaluation, and received Vitamin K1 (0.5 mg/kg i.m.) as prophylaxis, being thereafter excluded from this study. Among the neonate with NT greater than 20%, 426 babies have been considered, who had, at least, three evaluations in the first four days of life. 288 (68%) of the 426 newborns, had not important disease, while 138 (32%) were sick neonates; of these babies 88 (64%) had respiratory distress syndrome and 50 (36%) had an infectious condition. The mean of NT values of the 426 newborns decreased from the first (33.84%) to the 2nd day (32.72%), with a following increment in 3rd (35.29%) and 4th day of life (39.01%). Newborns with gestational age (G.A.) less than 34 weeks showed significantly lower values than newborns with G.A. between 34-37 weeks and those with G.A. greater than 37 weeks. No newborn with NT values greater than 20% either received vitamin K or showed symptoms of haemorrhagic disease in early or later neonatal period.

To investigate the effective usefulness of penicillin prophylaxis in splenectomized patients, we retrospectively focused on a group of sixty-two splenectomized patients affected by thalassemia major. Thirty-six out of 62 has been receiving monthly 1.200.000 Us. of benzathine-penicillin as prophylaxis. The remaining 26 did not receive prophylaxis, but was treated with antibacterial drugs as soon as symptoms of upper respiratory tract infection occurred. During a total period of eleven years of observation we did not observe any pneumococcal sepsis; the incidence of bacterial infections within the two groups is not different. We conclude for the uselessness of penicillin prophylaxis in splenectomized beta-thalassemic patients.

A new autosomally inherited dysfibrinogenaemia was recognized in 3 members of an Italian family. No bleeding tendency or thrombotic disease in any of the affected members were demonstrated. Coagulation tests revealed prolonged prothrombin, thrombin and Reptilase times. Plasma fibrinogen levels were normal with immunologic method and slightly reduced with chrono-metric assay: the other blood coagulation factors were normal. In addition, cross-immuno-electrophoresis performed on patients' plasma was indistinguishable from the normal. Dysfibrinogenaemia was confirmed by studying the purified fibrinogen. The fibrin polymerization curve, measured spectrophotometrically, showed a lower slope than the normal. A delay in fibrin monomer aggregation was revealed when compared to the normal at an equal concentration. The release of fibrinopeptides was normal. SDS polyacrylamide gel electrophoresis, isoelectric focusing and cross-immunoelectrophoresis of purified fibrinogen were not able to demonstrate any structural abnormality. The fibrinogen was named fibrinogen Genova.