Selenium binding to beef-kidney rhodanese.

The reaction of beef kidney rhodanese with selenosulfate was studied. The selenium-treated enzyme shows an absorption spectrum with a maximum at 375 nm attributable to a sulfoselenide group. This absorption is bleached by addition of cyanide. After cyanide treatment stoichiometric amount of selenocyanate can be found. The intrinsic fluorescence of rhodanese is quenched by addition of stoichiometric selenosulfate. This effect can be reversed by cyanide or sulfite but not by selenite or glutathione. By comparison with model complexes the selenium-rhodanese intermediate was identified as a cysteinyl-selenium derivative.     

Fingerprinting trifoliate orange germ plasm accessions with isozymes, RFLPs, and inter-simple sequence repeat markers

 Trifoliate orange [Poncirus trifoliata (L.) Raf.] is frequently used as a parent in citrus rootstock breeding, but the origin and amount of genetic diversity in germ plasm collections are poorly understood. Most accessions are self-compatible, but produce a mixture of sexual and apomictic seedlings. Variation among 48 vegetatively propagated trifoliate orange accessions was assessed at seven isozyme loci, together with the restriction fragment length polymorphisms (RFLPs) detected by 38 probe-enzyme combinations and the inter-simple sequence repeat (ISSR) markers generated by 11 primers. Isozymes and RFLPs detected few polymorphisms among accessions, although genetic analysis has shown that the common phenotype is heterozygous for four isozyme and at least four RFLP loci. ISSR amplification generated multiple banding profiles with an average of 58 fragments/primer/accession. These fragments were repeatable across DNA samples extracted from different trees of the same accession or extracted at different times, and across separate PCR runs. Seventeen unique marker phenotypes were identified. The 48 trifoliate orange accessions were classified into four major groups based on polymorphic ISSR markers. All large-flowered accessions are in group 4, while small-flowered accessions are in group 3. Many ISSR markers segregated in progeny derived by open-pollination (probably mostly selfing) of a common accession, indicating that these ISSR markers are also heterozygous. Accessions having identical genotypes for a large number of heterozygous markers are unlikely to have diverged by recombination. Thus the limited divergence we detected among most accessions most likely originated by mutation. ‘Monoembryonic’ and ‘Simmons’ differed from other accessions only in the loss of specific markers, indicating that they originated as zygotic seedlings of individuals similar to the common genotype. Three accessions recently introduced from China have relatively different fingerprints with 3–14 unique ISSR markers, and probably represent a much more divergent germ plasm that may be a valuable breeding resource. Received: 8 August 1996 / Accepted: 21 March 1997     

Preliminary study of the physical chemistry and catalytic properties of glutathione-S-transferase from human placenta

Glutathione-S-transferase activity has been identified in the cytosol of human placenta. The specific activity measured is about 50% of that found in human liver. While some kinetic data have a close correspondence with those attributed to transferases of other sources, the molecular weight (60.000 daltons) and electric properties of this protein are unusual. The inhibitory effect of several non-substrate compounds suggests that also the placental Glutathione-S-transferase may play some role in detoxication of exogenous substances.     

Nucleophosmin mutations in acute myeloid leukemia: A tale of protein unfolding and mislocalization

Nucleophosmin (NPM1) is an abundant, ubiquitously expressed protein mainly localized at nucleoli but continuously shuttling between nucleus and cytoplasm. NPM1 plays a role in several cellular functions, including ribosome biogenesis and export, centrosome duplication, chromatin remodeling, DNA repair, and response to stress stimuli. Much of the interest in this protein arises from its relevance in human malignancies. NPM1 is frequently overexpressed in solid tumors and is the target of several chromosomal translocations in hematologic neoplasms. Notably, NPM1 has been characterized as the most frequently mutated gene in acute myeloid leukemia (AML). Mutations alter the C‐terminal DNA‐binding domain of the protein and result in its aberrant nuclear export and stable cytosolic localization. In this review, we focus on the leukemia‐associated NPM1 C‐terminal domain and describe its structure, function, and the effect exerted by leukemic mutations. Finally, we discuss the possibility to target NPM1 for the treatment of cancer and, in particular, of AML patients with mutated NPM1 gene.     

Human glutathione transferase T2-2 discloses some evolutionary strategies for optimization of substrate binding to the active site of glutathione transferases

Rapid kinetic, spectroscopic, and potentiometric studies have been performed on human Theta class glutathione transferase T2-2 to dissect the mechanism of interaction of this enzyme with its natural substrate GSH. Theta class glutathione transferases are considered to be older than Alpha, Pi, and Mu classes in the evolutionary pathway. As in the more recently evolved GSTs, the activation of GSH in the human Theta enzyme proceeds by a forced deprotonation of the sulfhydryl group (pK(a) = 6.1). The thiol proton is released quantitatively in solution, but above pH 6.5, a protein residue acts as an internal base. Unlike Alpha, Mu, and Pi class isoenzymes, the GSH-binding mechanism occurs via a simple bimolecular reaction with k(on) and k(off) values at least hundred times lower (k(on) = (2.7 +/- 0.8) x 10(4) M(-1) s(-1), k(off) = 36 +/- 9 s(-1), at 37 degrees C). Replacement of Arg-107 by alanine, using site-directed mutagenesis, remarkably increases the pK(a) value of the bound GSH and modifies the substrate binding modality. Y107A mutant enzyme displays a mechanism and rate constants for GSH binding approaching those of Alpha, Mu, and Pi isoenzymes. Comparison of available crystallographic data for all these GSTs reveals an unexpected evolutionary trend in terms of flexibility, which provides a basis for understanding our experimental results.     

CD95/phosphorylated ezrin association underlies HIV-1 GP120/IL-2-induced susceptibility to CD95(APO-1/Fas)-mediated apoptosis of human resting CD4(+)T lymphocytes

CD95(APO-1/Fas)-mediated apoptosis of bystander uninfected T cells exerts a major role in the HIV-1-mediated CD4+ T-cell depletion. HIV-1 gp120 has a key role in the induction of sensitivity of human lymphocytes to CD95-mediated apoptosis through its interaction with the CD4 receptor. Recently, we have shown the importance of CD95/ezrin/actin association in CD95-mediated apoptosis. In this study, we explored the hypothesis that the gp120-mediated CD4 engagement could be involved in the induction of susceptibility of primary human T lymphocytes to CD95-mediated apoptosis through ezrin phosphorylation and ezrin-to-CD95 association. Here, we show that gp120/IL-2 combined stimuli, as well as the direct CD4 triggering, on human primary CD4(+)T lymphocytes induced an early and stable ezrin activation through phosphorylation, consistent with the induction of ezrin/CD95 association and susceptibility to CD95-mediated apoptosis. Our results provide a new mechanism through which HIV-1-gp120 may predispose resting CD4(+)T cell to bystander CD95-mediated apoptosis and support the key role of ezrin/CD95 linkage in regulating susceptibility to CD95-mediated apoptosis.