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.     

pp60c-src kinase activity in bovine coronary extracts is stimulated by ATP

pp60c-src kinase is believed to participate in regulating key cellular mechanisms including signal transduction and differentiation of smooth muscle during early embryogenesis. In this study, pp60c-src kinase activity was demonstrated in extracts from adult bovine coronary arterial smooth muscle. Activity, reflected by autophosphorylation of pp60c-src, phosphorylation of exogenous substrates, and phosphorylation of several endogenous substrates, was enhanced about 2 fold when added Mg2+ was replaced by Mn2+. Unexpectedly, activity was dramatically stimulated 20-50 fold by prior incubation with ATP. Such stimulation appears to be mediated through a novel mechanism which is independent of ATP-induced phosphorylation of reaction components. These new observations strongly suggest that a unique mechanism exists for regulation of coronary arterial pp60c-src kinase activity. Conceivably, this mechanism may serve important roles in modulating signal transduction and contractility of vascular smooth muscle.     

Spontaneously active and ATPMg-dependent protein phosphatase activity in vascular smooth muscle

A spontaneously active (Mr greater than 350,000) and an ATPMg-dependent phosphatase (Mr congruent to 140,000) were identified in bovine aortic smooth muscle. The spontaneously active phosphatase was effective in dephosphorylating both phosphorylase a (240nmol32P/min/mg) and phosphorylated myosin light chains (1000nmol32P/min/mg). In contrast, the ATPMg-dependent phosphatase was only effective in dephosphorylating phosphorylase a (400nmol32P/min/mg). Phosphorylase phosphatase activity of the ATPMg-dependent enzyme was suppressed by the well-characterized modulator protein (inhibitor-2), whereas the activity of the spontaneously active enzyme was unaffected. The aortic spontaneously active phosphatase did not convert to an ATPMg-dependent form when it was stored at 4 degrees or incubated at 30 degrees C in either the presence or absence of modulator protein. These findings suggest that spontaneous and ATPMg-dependent phosphatase activities described in these studies are probably ascribable to different enzymes. Since both phosphorylase and myosin light chains are phosphorylated when smooth muscle contracts these phosphatases may participate in coordinating arterial contractility and metabolism.     

Escherichia coli protein StpA stimulates self-splicing by promoting RNA assembly in vitro.

An Escherichia coli gene, stpA, has been identified and cloned based on its ability to suppress the Td- phenotype of a resident, splicing-defective phage T4 td (thymidylate synthase) gene. The stpA gene, which was localized to 60.24 min on the E. coli chromosome, encodes a 15.3-kDa protein. Overproduction of StpA in vivo led to an increase in td pre-mRNA levels and modest enhancement of td mRNA:pre-mRNA ratios. Consistent with its in vivo effect, purified StpA promoted RNA splicing in vitro, and facilitated RNA annealing and strand exchange with model substrates. These results suggest that StpA promotes splicing of the intron by binding RNA nonspecifically, resolving misfolded precursor molecules and facilitating association of critical base pair elements. Furthermore, proteinase K treatment of StpA-assembled precursors prior to the initiation of the splicing reaction still resulted in splicing enhancement, indicating that StpA is not required for the catalytic step, unlike the Neurospora splicing effector CYT-18, whose presence was necessary for catalysis to proceed. Together these results suggest that StpA has chaperone activity in vitro, with the property of promoting assembly of the precursors into an active conformation, in contrast to splicing effectors that stabilize the catalytically active intron structure.     

Blood eosinophils and serum eosinophil cationic protein in patients with acute and chronic urticaria

We have analysed the relationship of blood eosinophil count and serum eosinophil cationic protein (ECP) levels in patients with acute and chronic idiopathic urticaria. The ECP levels and eosinophil counts were measured in the peripheral blood of 15 patients with acute urticaria, 25 with chronic idiopathic urticaria and 10 normal healthy subjects. Blood eosinophil counts and serum ECP levels increased in all patients with acute urticaria. Concerning patients affected by chronic urticaria, taking into account the recrudescence of the disease at the moment of taking the blood sample, only symptomatic patients showed increased eosinophil blood values whereas serum ECP levels were increased both in symptomatic and asymptomatic patients. Furthermore, serum ECP levels in chronic urticaria did not correlate with the peripheral eosinophil counts, as they did in acute urticaria. The results of the present study indicate that eosinophils may play a role in the inflammatory mechanisms in patients with acute and chronic urticaria showing a positive correlation between serum ECP levels and disease activity.     

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.     

Purification of cytoplasmic antigens from the mycelial phase of Candida albicans: Possible advantages of its use in Candida serology

The serology of candidiasis is complicated by the use of poorly defined antigens. Total extracts of the yeast phase have been commonly used as cytoplasmic antigen, without regard to the significant amounts of carbohydrate that may contaminate such preparations. This is particularly true in the case of commercially available antigens that have been used as cytoplasmic antigens but actually are richer in carbohydrate than in protein. Affinity chromatography in concanavalin A — Sepharose provides a simple procedure to separate carbohydrates, mainly mannan, from protein antigens in whole Candida extracts. By using mannan-poor antigens, the specificity of serological reactions can be increased considerably, since both the positive reactions seen in asymptomatic donors and the cross-reactions seen in patients infected with other fungi are due to anti-mannan antibodies. In contrast, both anti-mannan and anti-cytoplasmic antigen antibodies can be detected in patients suspected of systemic candidiasis. On the other hand, absolute specificity may never be achieved for systemic candidiasis. We have found antibodies against cytoplasmic antigen in a patient allergic to C. albicans, in whom the microorganism was isolated from fecal material. It appears that, under favorable conditions, mucosal sensitization may also trigger a systemic reaction directed against both mannan and cytoplasmic antigens.Publication no. 341 from The Department of Basic and Clinical Immunology and Microbiology, Medical University of South Carolina.