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.     

Regression of human melanoma xenografts in nude mice injected with methotrexate linked to monoclonal antibody 225.28 to human high molecular weight-melanoma associated antigen

Summary Intravenous injections into nude mice of 5 mg/kg methotrexate (MTX) linked to the antibody to human high molecular weight-melanoma associated antigen (HMW-MAA), monoclonal antibody (mAb) 225.28, an IgG_2a, on days 1, 4, 7, 10 and 14, starting 24 h after subcutaneous inoculation of 2 × 10⁶ cultured human M21 melanoma cells inhibited mean tumor volume by 90% on day 14 and by 65% on day 50 after the beginning of the treatment. Injections of equimolar amounts of free MTX and MTX linked to normal mouse IgG or to an isotypematched myeloma protein did not inhibit tumor growth significantly. MTX linked to mAb 225.28 did not inhibit the xenograft of a subline of human melanoma cell line M21 without detectable expression of HMW-MAA. In a clonogenic assay, the MTX-225.28 conjugate was three times more potent in inhibiting the growth of M21 melanoma cells than free MTX, but did not inhibit the growth of kidney carcinoma cells Caki-1, which do not express high-M _r MAA. In contrast, MTX linked to the mAb DAL K29, reacting with kidney carcinoma cells Caki-1, inhibited their growth but did not affect that of melanoma cells. M21 melanoma cells isolated from the residual tumor of a mouse treated with the MTX-225.28 conjugate did not differ in their reactivity with mAb 225.28 and in their sensitivity to MTX when compared with M21 cells from an untreated mouse.     

DQ α and β RFLP reveals the composition of the DQ molecule recognized by T-cell clones

Pst I RFLP, revealed with DQ and DQ probes, was compared with Taq I RFLP using a panel of DR-homozygous cell lines and HLA-typed family members. Taq I patterns, characteristic for each DR-associated DQ and allelic forms, were recognized in the homozygous state and then proven to segregate in the heterozygous members of informative families. The presence of both specific and chains was found to be necessary to form the type of DQ molecule specifically recognized by two alloreactive T-cell clones. Particular and associations also seem to be responsible for some Dw splits of the DRw6-positive cells. Taq I RFLP analysis may be more complex than the Pst I analysis, but is certainly more informative and complete, considering the type of information we were seeking by performing these types of experiments.Abbreviations used in this paper BSA bovine serum albumin - GLO glyoxalase - kb kilobase(s) - LCL lymphoblastoid cell line - MHC major histocompatibility complex - PBL peripheral blood lymphocyte - PLT primed lymphocyte test - RFLP restriction fragment length polymorphism - SDS sodium dodecyl sulfate - SSC standard sodium citrate - SSCP sodium, sodium citrate, sodium phosphate - TBE Tris-borate, boric acid, ethylenediaminetetraacetate (EDTA) - TCGF T-cell growth factor     

EBNA-negative polyclonal B cells derived from a long-term culture of unclassified acute lymphoblastic leukemia: IL-2-like activity of culture's supernatant

A long-term culture of bone marrow lymphoblasts in a case of unclassified acute lymphoblastic leukemia is described. Cells lacking any lymphocytic marker in the early phase of the culture were gradually substituted by B cells showing a pattern of polyclonality. The culture supernatant contained high levels of immunoglobulins also showing interleukin 2 activity. Search for antigens related to the Epstein-Barr virus was negative. A clonal expansion of B cells versus spontaneous differentiation of unclassified leukemic cells is discussed; the long-term culture technique as a tool for a better evaluation of leukemic cells is suggested and discussed.     

NMR studies in solution of poly-L-proline

The isomerization of poly-L -proline in different solvents has been studied by NMR spectroscopy. Different resonance signals for the CH_α protons have been obtained for the two different helical conformations of thus compound, namely form I and form II.     

Pig kidney dopa decarboxylase: inactivation by iodoacetamide and sequence of the carboxyamidomethylcysteine-containing peptide

Pig kidney 3,4-dihydroxyphenylalanine (Dopa) decarboxylase is inactivated by iodoacetamide following pseudo-first order reaction kinetics. The apparent first order rate constant for inactivation is proportional to the concentration of iodoacetamide and a second order rate constant of 37 M-1 min-1 is obtained at pH 6.8 and 25 degrees C. Cyanogen bromide fragmentation of iodo(1-14C)acetamide - modified inactivated Dopa decarboxylase followed by trypsin digestion yields a single radioactive peptide. Automated Edman degradation reveals a heptapeptide sequence which contains labeled carboxyamidomethylcysteine. This finding and the results of the incorporation of the label from ido (1-14C)acetamide into the enzyme clearly indicate that the modification of 1 mol of SH per mol of enzyme dimer is responsible for the inactivation process. The labeled peptide, which was located by means of limited proteolysis on the fragment corresponding to the COOH-terminal third of the enzyme, has been aligned with a 7 amino acid stretch of Drosophila enzyme. Although this region appears highly conserved in the Dopa decarboxylase enzymes, the cysteinyl residue is not conserved. This observation together with the spectral binding properties of the iodoacetamide inactivated enzyme argue against a functional role for the modifiable cysteine in the mechanism of action of pig kidney enzyme. It is suggested that the loss of pig kidney decarboxylase activity produced by iodoacetamide modification might be attributable to steric hindrance. This could be due to the presence of the bulky acetamidic group on a cysteine residue at, or near, the active center or in a site of strategic importance to the maintenance of the active site topography.     

The extension reached by the minimization of the polarity distances during the evolution of the genetic code

Summary The level reached by the optimization of the polarity distances during the evolution of the genetic code was investigated. The results, although not conclusive, indicate that this optimization level is higher than the data reported in the literature. The results seem compatible with the reaching of an evolutionary minimum, with respect to the optimization of the polarity distances, by the genetic code during its formation.     

Islet-Activating Protein Inhibits the β-Adrenergic Receptor Facilitation Elicited by γ-Aminobutyric AcidB Receptors

gamma-Aminobutyric acidB (GABAB) receptor recognition sites that inhibit cyclic AMP formation, open potassium channels, and close calcium channels are coupled to these effector systems by guanine nucleotide binding proteins (G proteins). These G proteins are ADP-ribosylated by islet-activating protein (IAP), also known as pertussis toxin. This process prevents receptor coupling to these G proteins. In slices of cerebral cortex and hippocampus from rat, stimulation of GABAB receptors with baclofen, a receptor agonist, also potentiates the accumulation of cyclic AMP stimulated by beta-adrenergic agonists. It was unknown whether those GABAB receptors that potentiate the beta-adrenergic response were also sensitive to IAP. IAP was injected intracerebroventricularly into rats to ADP-ribosylate IAP-sensitive G proteins. Four days after the IAP injection, 38% and 52% of these G proteins from cerebral cortex and hippocampus, respectively, were ADP-ribosylated by the IAP injection. In slices of both structures prepared from IAP-treated rats, the GABAB receptor-mediated potentiation of the beta-adrenergic receptor response was attenuated. Thus, many GABAB receptor-mediated responses are coupled to IAP-sensitive G proteins.     

Changes in Cyclins and Cyclin-Dependent Kinases Induced by DNA Damaging Agents in a Human Ovarian Cancer Cell Line Expressing Mutated or Wild-Type P53

The expression of cyclins, cyclin-dependent kinases (cdk), and cdk inhibitors was evaluated in clones from a human ovarian cancer cell line transfected with a temperature-sensitive mutant of p53, after treatment with the anticancer agents doxorubicin (DX) and AMSA. The two drugs were selected on the basis of their activity in these clones, since AMSA is equally active in cells expressing mutated or wild-type (wt) p53, while DX was much less cytotoxic in cells expressing wt p53. In untreated cells, the expression of wt p53 induced an accumulation of cells in the G2 and perhaps also the G1 phase of the cell cycle. Concomitantly cyclin B1 and cdc2 increased. Cyclin E and particularly D1 levels were also raised by wt p53 expression. Treatment of mutated p53-expressing cells (SK23a cells kept at 37°C) with DX or, more so, with AMSA, resulted in a strong accumulation of cyclin B1 and cdc2, in accordance with their ability to block cells in G2 phase of the cell cycle. Wt p53-expressing cells (SK23a cells kept at 32°C) treated with the drugs showed an increase in p21 expression and consequently decreased kinase activity after immunoprecipitation with p21 antibodies. Cdc2-associated kinase activity was also reduced in these conditions. We could also observe a decrease in the percentage of cells in G1 and G2 phases and an accumulation of cells in S phase after both DX and AMSA. Cdk2, retinoblastoma, and p27 levels did not change significantly. Treatment with DX or AMSA caused similar effects, suggesting that p53-induced changes in cyclin, cdk, and cdk inhibitors after DNA damage are not responsible for the marked reduction in the cytotoxicity of DX we observed in wt p53-expressing cells.     

Spermatophore development and sperm ultrastructure inCraterostigmus tasmanianus (Chilopoda,Craterostigmomorpha)

 Spermatophore development and ultrastructure of the mature sperm of Craterostigmus tasmanianus were studied using light and electron microscopy. In C. tasmanianus, as in the Scolopendromorpha, the spermatophore develops within the vas deferens. The latter consists of three parts, each with a different morphology. The first may be involved in guiding the sperm to roll up into typical ring-like structures, while the other two, which show an evident secretory activity, secrete the acellular wall of the spermatophores. The ultrastructure of mature spermatozoa showed that a very close similarity exists between Craterostigmomorpha and Lithobiomorpha, especially regarding the organization of the connecting piece. Based on this similarity, we consider the Craterostigmomorpha together with the Scolopendromorpha, Geophilomorpha and Lithobiomorpha (=Pleurostigmophora) to be the sister group of the Scutigeromorpha. Accepted: 2 June 1996