Analysis of X-chromosome inactivation and presumptive expression of the Wiskott-Aldrich syndrome (WAS) gene in hematopoietic cell lineages of a thrombocytopenic carrier female of WAS

Summary We report on a thrombocytopenic female belonging to a pedigree with the Wiskott-Aldrich syndrome (WAS). Restriction fragment length polymorphism (RFLP) analysis with probe M27, closely linked to the WAS gene, demonstrated that she is a carrier of WAS. Both small-sized and normal-sized platelets were present, suggesting that, unlike the vast majority of WAS carriers, she does not manifest nonrandom X-chromosome inactivation in the thrombopoietic cell lineage. Study of X-chromosome inactivation by means of RFLP and methylation analysis demonstrated that the pattern of X-chromosome inactivation was nonrandom in T lymphocytes, but random in granulocytes. While this is the first complete report on the occurrence of thrombocytopenia in a carrier female of WAS as the result of atypical lyonization, it also suggests that expression of the WAS gene occurs at (or extends up to) a later stage than the multipotent stem cell along the hematopoietic differentiation pathway.     

Purification and properties of DNA topoisomerase II from Daucus carota cells

Topoisomerase II was partially purified from Daucus carota cellsby a procedure including ammonium sulphate fractionation, ion-exchange,and affinity chromatography steps. The type II enzyme, identifiedfor its ability to unknot knotted P4 DNA and decatenate Trypanosomacruzi kDNA, requires ATP and Mg²⁺ for activity. The unknottingactivity was sensitive to an inhibitor of the mammalian typeII enzyme, the drug VP16 (IC₅₀ 32 mmol m^–3), whereas inhibitorsof DNA gyrase showed a limited effect on activity. The SDS-PAGEanalysis of the dsDNA cellulose fraction revealed the presenceof four polypeptides of apparent molecular masses of 72, 71,34, and 33 kDa among which only a polypeptide of about 70 kDacrossreacted with antibodies against yeast topoisomerase II.Immunoprecipitation experiments with monoclonal antibodies tothe and ß isoforms of the human enzyme confirmedthe recognition of a polypeptide of 70 kDa. The sedimentationcoefficient (S) of the topoisomerase II in the phosphocellulosefraction, calculated by analytical glycerol gradient, was 6.1corresponding to a molecular mass of about 123 kDa. Resultssuggest the presence in carrot of a protein of molecular massof 70 kDa having the typical properties of an eukaryotic topoisomeraseII and carrying epitopes recognized by MoAbs to both human and ß enzymes. The 70 kDa polypeptide might then representthe monomer of a homodimer enzyme of 123 kDa. Key words: Daucus carota, topoisomerase II, immunoprecipitation     

Characterization of a Small Family (CAIII) of Microsatellite-Containing Sequences with X-Y Homology

Four X-linked loci showing homology with a previously described Y-linked polymorphic locus (DYS413) were identified and characterized. By fluorescent in situ hybridization (FISH), somatic cell hybrids, and YAC screening, the X-linked members of this small family of sequences (CAIII) all map in Xp22, while the Y members map in Yq11. These loci contribute to the overall similarity of the two genomic regions. All of the CAIII loci contain an internal microsatellite of the (CA)_n type. The microsatellites display extensive length polymorphism in two of the X-linked members as well as in the Y members. In addition, common sequence variants are found in the portions flanking the microsatellites in two of the X-linked members. Our results indicate that, during the evolution of this family, length variation on the Y chromosome was accumulated at a rate not slower than that on the X chromosome. Finally, these sequences represent a model system with which to analyze human populations for similar X- and Y-linked polymorphisms. Received: 29 July 1996 / Accepted: 15 January 1997     

Simultaneous analysis of substrates,products, and inhibitors of xanthine oxidase by high-pressure liquid chromatography and gas chromatography

Procedures are presented for the simultaneous analysis of hypoxanthine, xanthine, allopurinol, oxipurinol, and uric acid in standard mixtures and physiological fluids using gas chromatography (gc) or high-pressure liquid chromatography (hplc). Excellent correlation was obtained between the two methods for hypoxanthine, xanthine, oxipurinol, and uric acid. There are advantages and disadvantages to both methods. hplc requires no prior derivatization, uses isocratic elution with a buffer containing no organic solvent, and has 50- to 100-fold greater sensitivity than gc. Simpler methods of prepurification, readily adapted to clinical laboratories, can be used for hplc analysis. Although substances that are found in some urine samples from cancer patients interfere with hplc, separations by gc are not affected by these substances.     

Hydrolysis of cyclosporin A: Identification of 1,11 seco-cyclosporin A and 4,5 secoisoCyclosporin A by FAB-MS/MS

We have previously reported that treatment of CsA with aqueous HCl gives rise to the formation of a number of water-soluble compounds. Two of these were identified from their FAB-MS/MS spectra as open-chain nona- and decapeptides. We describe here the identification of two other main compounds deriving from the same treatment. Identification was rendered possible from the comparison of their FAB-MS/MS spectra with those of methyl and acetyl derivatives. The two compounds are water-soluble, open-chain undecapeptides corresponding to 1,11 seco-CsA and of 4,5 seco-isoCsA, respectively.     

Correction to: The evolution of life cycle assessment in european policies over three decades

The International Journal of Life Cycle Assessment -     

Hidden Behind Autophagy: The Unconventional Roles of ATG Proteins

Macroautophagy (hereafter referred to as autophagy) is an evolutionarily conserved intracellular catabolic transport route that generally allows the lysosomal degradation of cytoplasmic components, including bulk cytosol, protein aggregates, damaged or superfluous organelles and invading microbes. Target structures are sequestered by double‐membrane vesicles called autophagosomes, which are formed through the concerted action of the autophagy (ATG)‐related proteins. Until recently it was assumed that ATG proteins were exclusively involved in autophagy. A growing number of studies, however, have attributed functions to some of them that are distinct from their classical role in autophagosome biogenesis. Autophagy‐independent roles of the ATG proteins include the maintenance of cellular homeostasis and resistance to pathogens. For example, they assist and enhance the turnover of dead cells and microbes upon their phagocytic engulfment, and inhibit murine norovirus replication. Moreover, bone resorption by osteoclasts, innate immune regulation triggered by cytoplasmic DNA and the ER‐associated degradation regulation all have in common the requirement of a subset of ATG proteins. Microorganisms such as coronaviruses, Chlamydia trachomatis or Brucella abortus have even evolved ways to manipulate autophagy‐independent functions of ATG proteins in order to ensure the completion of their intracellular life cycle. Taken together these novel mechanisms add to the repertoire of functions and extend the number of cellular processes involving the ATG proteins.     

Obstructive lung diseases and inhaler treatment: results from a national public pragmatic survey

Background

The opinions held by the general population on obstructive lung disease and inhaler devices could influence asthma and chronic obstructive pulmonary disorder (COPD) management and treatment adherence.The aim of the present public pragmatic survey was to evaluate the opinions, beliefs and perceptions of Italian people with respect to respiratory diseases as well as their perspectives on the use of inhaler devices.

Methods

This survey was conducted on a group of 2,008 individuals forming a representative sample of the Italian population aged 15 years and over. It was based on personal interviews that were administered in the homes of the respondents using a structured questionnaire that took approximately 30 minutes.

Results

Awareness of obstructive lung diseases is poor. Asthma, but not COPD, was perceived as a common and increasingly prevalent disease by the majority of the interviewees. Allergy, pollution and smoking were considered to be responsible for both of these diseases. The rates at which the respondents claimed to be suffering from asthma and COPD were lower than expected (4% and 2%, respectively). Inhaled drugs were recognised as the main treatment by 65% of the respondents. The great majority of respondents attributed positive characteristics to the inhaler device (e.g., safety, reliability, effectiveness, ease of use and practicality). Compared to people who have never used inhaler devices, individuals who suffer from asthma or COPD were more confident in their use and showed a greater belief in their safety, reliability and trustworthiness. People older than 64 years showed less attention to the properties of these devices.

Conclusions

The present results highlight the need for public interventions aimed at improving awareness of obstructive lung disease and reveal various potentialities and critical issues for inhaler device usage. Switching of devices was considered feasible by most of the interviewees, as long as the choice is carefully explained by their physician.