Identification of critical, conserved vicinal aspartate residues in mammalian and bacterial ADP-ribosylarginine hydrolases.

NAD:arginine ADP-ribosyltransferases and ADP-ribosylarginine hydrolases catalyze opposing arms of a putative ADP-ribosylation cycle. ADP-ribosylarginine hydrolases from mammalian tissues and Rhodospirillum rubrum exhibit three regions of similarity in deduced amino acid sequence. We postulated that amino acids in these consensus regions could be critical for hydrolase function. To test this hypothesis, hydrolase, cloned from rat brain, was expressed as a glutathione S-transferase fusion protein in Escherichia coli and purified by glutathione-Sepharose affinity chromatography. Conserved amino acids in each of these regions were altered by site-directed mutagenesis. Replacement of Asp-60 or Asp-61 with Ala, Gln, or Asn, but not Glu, significantly reduced enzyme activity. The double Asp-60 --> Glu/Asp-61 --> Glu mutant was inactive, as were Asp-60 --> Gln/Asp-61 --> Gln or Asp-60 --> Asn/Asp-61 --> Asn. The catalytically inactive single and double mutants appeared to retain conformation, since they bound ADP-ribose, a substrate analogue and an inhibitor of enzyme activity, with affinity similar to that of the wild-type hydrolase and with the expected stoichiometry of one. Replacing His-65, Arg-139, Asp-285, which are also located in the conserved regions, with alanine did not change specific activity. These data clearly show that the conserved vicinal aspartates 60 and 61 in rat ADP-ribosylarginine hydrolase are critical for catalytic activity, but not for high affinity binding of the substrate analogue, ADP-ribose.

Characterization of NAD:arginine ADP-ribosyltransferases.

NAD:arginine mono-ADP-ribosyltransferases catalyze the transfer of ADP-ribose from NAD to the guanidino group of arginine on a target protein. Deduced amino acid sequences of one family (ART1) of mammalian ADP-ribosyltransferases, cloned from muscle and lymphocytes, show hydrophobic amino and carboxyl termini consistent with glycosylphosphatidylinositol (GPI)-anchored proteins. The proteins, overexpressed in mammalian cells transfected with the transferase cDNAs, are released from the cell surface with phosphatidylinositol-specific phospholipase C (PI-PLC), and display immunological and biochemical characteristics consistent with a cell surface, GPI-anchored protein. In contrast, the deduced amino acid sequence of a second family (ART5) of transferases, cloned from murine lymphoma cells and expressed in high abundance in testis, displays a hydrophobic amino terminus, consistent with a signal sequence, but lacks a hydrophobic signal sequence at its carboxyl terminus, suggesting that the protein is destined for export. Consistent with the surface localization of the GPI-linked transferases, multiple surface substrates have been identified in myotubes and activated lymphocytes, and, notably, include integrin alpha subunits. Similar to the bacterial toxin ADP-ribosyltransferases, the mammalian transferases contain the characteristic domains involved in NAD binding and ADP-ribose transfer, including a highly acidic region near the carboxy terminus, which, when disrupted by in vitro mutagenesis, results in a loss of enzymatic activity. The carboxyl half of the protein, synthesized as a fusion protein in E. coli, possessed NADase, but not ADP-ribosyltransferase activity. These findings are consistent with the existence at the carboxyl terminus of ART1 of a catalytically active domain, capable of hydrolyzing NAD, but not of transferring ADP-ribose to a guanidino acceptor.

Application of hematopoietic growth factors (G-CSF and GM-CSF) in the treatment of chemotherapy-induced or idiopathic bone marrow failure.

Fifteen patients with various myeloid and lymphoid neoplasias after receiving highly myelotoxic chemotherapy were treated with a single daily dose of 5 micrograms/kg of GM-CSF. G-CSF at a daily dose of 5-8 micrograms/kg was used in four patients with severe pancytopenia in the course of acute or chronic lymphoid leukemia treated with cytotoxic agents, and in two patients with idiopathic aplastic anemia. The administration of cytokines was provided for 4-10 days GM-CSF increased the WBC in twelve out of fifteen patients, mainly because of an increase in the number of neutrophils. Six patients receiving GM-CSF demonstrated rapid platelet recoveries. The rapid increase of the WBC was observed in all G-CSF treated patients. In one patient with aplastic anemia the WBC/ANC decreased rapidly to the initial values after the cessation of the G-CSF therapy. Fast platelet recovery was seen in three patients treated with G-CSF.

[Intermediate lymphocytic lymphoma]. / Chloniaki o posrednim stopniu zróznicowania (Intermediate Lymphocytic Lymphoma, ILL).

The characteristic features are presented of intermediate lymphocytic lymphomas which have suggested the pathomorphological and clinical peculiarity of this disease entity. They occur in two forms: diffuse and nodular which are two phases in the development of the disease. The phenotypic and karyotypic characteristics of these lymphomas resemble those of chronic lymphocytic leukaemia, but their more malignant course is similar to that of centrocytic lymphomas.