On the correct use of ‘data’

Acknowledgement  Thanks are due to Drs. Paul Johnston (P.Johnston@ exeter.ac.uk) and E. William Beese (ebeese@t-online.de) for kindly providing dictionary definitions and for sharing their thoughts about the correct use of ‘data’.     

Thrombosis in children with hematologic malignancies

This review is based on pediatric reports (- January 2004) on the presence of symptomatic thrombosis in children with hematologic malignancies, mainly acute lymphoblastic leukemia, treated with different treatment protocols and associated with acquired and inherited prothrombotic risk factors (factor V G1691A, factor G20210A, MTHFR C677T genotypes, protein C, protein S, antithrombin, elevated levels of lipoprotein(a), and homocysteine). The interactions of treatment modalities, study designs, ethnical backgrounds and associated central lines are discussed. Based on the data presented here, we suggest the use of prednisone and E. coli asparaginase concomitantly administered in a leukemic patient suffering a prothrombotic risk factor to be responsible for the onset of venous thrombosis in the majority of cases. In addition, primary preventive anticoagulant/antithrombotic strategies are discussed.     

Synthesis, structural investigations and biological evaluation of novel hexahydropyridazine-1-carboximidamides, -carbothioamides and -carbothioimidic acid esters as inducible nitric oxide synthase inhibitors

Local excess of nitric oxide (NO) has been implicated in beta-cell damage, thus, a possible approach to the treatment of autoimmune IDDM is the selective inhibition of inducible nitric oxide synthase (iNOS). A series of variously substituted hexahydropyridazine-1-carbothioamides, -carbothioimidic acid esters and -carboximidamides was synthesized and dose-dependently evaluated as potential inhibitors of iNOS. The screening of the title compounds was performed with insulin-producing RIN-5AH cells and a combination of IL1-1 beta and IFN-gamma as inducers of cellular NO production. The structure-activity analysis revealed that the variation of substituents in the position 1 of the hexahydropyridazine strongly influences the inhibitory activity to iNOS as well as being critical for RIN cell survival. Among the compounds tested, the hexahydropyridazine-1-carbothioamides showed particularly significant inhibitory effects. However, for an efficient iNOS inhibition substitution at the nitrogen of the 1-carbothioamide group is important. Thus, the introduction of aliphatic chains such as propyl or butyl and of cyclic moieties such as cyclohexyl, 3-methoxyphenyl, and 4-methoxyphenyl (IC(50): 0.5-2.1 mM), respectively, provided compounds with similar inhibitory activity to aminoguanidine (IC(50): 0.3 mM), a common standard substance used for the selective inhibition of iNOS. However, the 1-carboximidamides, which represent more structurally related semicyclic derivatives of aminoguanidine, caused only incomplete iNOS inhibition. The hexahydropyridazine-1-carbothioimidic acid esters caused dose- and substituent-dependent damage of RIN-5AH cells. The toxicity of the synthesized compounds increased markedly if aliphatic substituents at the exocyclic N atom(s) were replaced by variously substituted aromatic rings.     

Prostasin, a membrane-anchored serine peptidase, regulates sodium currents in JME/CF15 cells, a cystic fibrosis airway epithelial cell line

Prostasin is a tryptic peptidase expressed in prostate, kidney, lung, and airway. Mammalian prostasins are related to Xenopus channel-activating protease, which stimulates epithelial Na+ channel (ENaC) activity in frogs. In human epithelia, prostasin is one of several membrane peptidases proposed to regulate ENaC. This study tests the hypothesis that prostasin can regulate ENaC in cystic fibrosis epithelia in which excessive Na+ uptake contributes to salt and water imbalance. We show that prostasin mRNA and protein are strongly expressed by human airway epithelial cell lines, including immortalized JME/CF15 nasal epithelial cells homozygous for the DeltaF508 cystic fibrosis mutation. Epithelial cells transfected with vectors encoding recombinant soluble prostasin secrete active, tryptic peptidase that is highly sensitive to inactivation by aprotinin. When studied as monolayers in Ussing chambers, JME/CF15 cells exhibit amiloride-sensitive, transepithelial Na+ currents that are markedly diminished by aprotinin, suggesting regulation by serine-class peptidases. Overproduction of membrane-anchored prostasin in transfected JME/CF15 cells does not augment Na+ currents, and trypsin-induced increases are small, suggesting that baseline serine peptidase-dependent ENaC activation is maximal in these cells. To probe prostasin's involvement in basal ENaC activity, we silenced expression of prostasin using short interfering RNA targeting of prostasin mRNA's 3'-untranslated region. This drops ENaC currents to 26 +/- 9% of baseline. These data predict that prostasin is a major regulator of ENaC-mediated Na+ current in DeltaF508 cystic fibrosis epithelia and suggest that airway prostasin is a target for therapeutic inhibition to normalize ion current in cystic fibrosis airway.     

Mitogenic effects of phospholipase D and phosphatidic acid in transiently permeabilized astrocytes: effects of ethanol

Investigations of lipid-mediated signalling pathways are often limited by a lack of methods for the intracellular delivery of lipid messengers. We established a procedure for the transient permeabilization of astrocytes by an oxygen-insensitive mutant of streptolysin-O (SLO) to investigate the participation of the phospholipase D (PLD) signalling pathway in astroglial cell proliferation. Exogenous PLD, when incubated in the presence of SLO, caused an increase in DNA synthesis (measured by thymidine incorporation) which was completely suppressed by ethanol (0.3%, v/v). In parallel experiments, phosphatidic acid also induced a dose-dependent mitogenic response which, however, was not affected by the presence of ethanol. Phosphatidic acid was more effective in this assay than diacylglycerol but its effect was sensitive to the protein kinase inhibitor Ro 31-8220. Our findings provide direct evidence that disruption of the PLD signalling pathway by ethanol is sufficient to suppress astroglial proliferation, an effect that might contribute to the inhibition of brain growth in alcoholic embryopathy.     

Acid secretion and proton conductance in human airway epithelium

Acid secretion and proton conductive pathways across primary human airway surface epithelial cultures were investigated with the pH stat method in Ussing chambers and by single cell patch clamping. Cultures showed a basal proton secretion of 0.17 +/- 0.04 micromol.h(-1).cm(-2), and mucosal pH equilibrated at 6.85 +/- 0.26. Addition of histamine or ATP to the mucosal medium increased proton secretion by 0.27 +/- 0.09 and 0.24 +/- 0.09 micromol.h(-1).cm(-2), respectively. Addition of mast cells to the mucosal medium of airway cultures similarly activated proton secretion. Stimulated proton secretion was similar in cultures bathed mucosally with either NaCl Ringer or ion-free mannitol solutions. Proton secretion was potently blocked by mucosal ZnCl(2) and was unaffected by mucosal bafilomycin A(1), Sch-28080, or ouabain. Mucosal amiloride blocked proton secretion in tissues that showed large amiloride-sensitive potentials. Proton secretion was sensitive to the application of transepithelial current and showed outward rectification. In whole cell patch-clamp recordings a strongly outward-rectifying, zinc-sensitive, depolarization-activated proton conductance was identified with an average chord conductance of 9.2 +/- 3.8 pS/pF (at 0 mV and a pH 5.3-to-pH 7.3 gradient). We suggest that inflammatory processes activate proton secretion by the airway epithelium and acidify the airway surface liquid.     

ATP-dependent remodeling of the spliceosome: intragenic suppressors of release-defective mutants of Saccharomyces cerevisiae Prp22

The essential splicing factor Prp22 is a DEAH-box helicase that catalyzes the release of mRNA from the spliceosome. ATP hydrolysis by Prp22 is necessary but not sufficient for spliceosome disassembly. Previous work showed that mutations in motif III (635SAT637) of Prp22 that uncouple ATP hydrolysis from spliceosome disassembly lead to severe cold-sensitive (cs) growth defects and to impaired RNA unwinding activity in vitro. The cs phenotype of S635A (635AAT) can be suppressed by intragenic mutations that restore RNA unwinding. We now report the isolation and characterization of new intragenic mutations that suppress the cold-sensitive growth phenotypes of the T637A motif III mutation (SAA), the H606A mutation in the DEAH-box (DEAA), and the R805A mutation in motif VI (804QAKGRAGR811). Whereas the T637A and H606A proteins are deficient in releasing mRNA from the spliceosome at nonpermissive temperature in vitro, the suppressor proteins have recovered mRNA release activity. To address the mechanisms of suppression, we tested ATPase and helicase activities of Prp22 suppressor mutant proteins and found that the ability to unwind a 25-bp RNA duplex was not restored in every case. This finding suggests that release of mRNA from the spliceosome is less demanding than unwinding of a 25-bp duplex RNA; the latter reaction presumably reflects the result of several successive cycles of ATP binding, hydrolysis, and unwinding. Increasing the reaction temperature allows H606A and T637A to effect mRNA release in vitro, but does not restore RNA unwinding by T637A.     

Characterization of dominant-negative mutants of the DEAH-box splicing factors Prp22 and Prp16

Saccharomyces cerevisiae Prp22 and Prp16 are RNA-dependent ATPases required for pre-mRNA splicing. Both proteins are members of the DEXH-box family of nucleic acid-dependent NTPases. Prior mutational analysis of Prp22 and Prp16 identified residues within conserved motifs I (GXGKT), II (DEAH), and VI (QRXGRXGR) that are required for their biological activity. Nonfunctional Prp22 and Prp16 mutants exerted a dominant negative effect on cell growth. Here we show that overexpression of lethal Prp22 mutants leads to accumulation of unspliced pre-mRNAs and excised introns in vivo. The biochemical basis for the lethality and inhibition of splicing in vivo was determined by purifying and characterizing recombinant mutant proteins. The lethal Prp22 mutants D603A and E604A in motif II and Q804A and R808A in motif VI were defective for ATP hydrolysis and mRNA release from the spliceosome, but were active in promoting step 2 transesterification. Lethal Prp16 mutants G378A and K379A in motif I; D473A and E474A in motif II; and Q685A, G688A, R689A, and R692A in motif VI were defective for ATP hydrolysis and step 2 transesterification chemistry. The ATPase-defective mutants of Prp16 and Prp22 bound to spliceosomes in vitro and blocked the function of the respective wild-type proteins in trans. Comparing the mutational effects in Prp16 and Prp22 highlights common as well as distinct structural requirements for the ATP-dependent steps in pre-mRNA splicing.     

A giant protease with a twist: the TPP II complex from Drosophila studied by electron microscopy

Tripeptidyl peptidase II (TPP II) is an exopeptidase of the subtilisin type of serine proteases that is thought to act downstream of the proteasome in the ubiquitin-proteasome pathway. Recently, a key role in a pathway parallel to the ubiquitin-proteasome pathway has been ascribed to TPP II, which forms a giant protease complex in mammalian cells. Here, we report the 900-fold purification of TPP II from Drosophila eggs and demonstrate via cryo-electron microscopy that TPP II from Drosophila melanogaster also forms a giant protease complex. The presented three-dimensional reconstruction of the 57 x 27 nm TPP II complex at 3.3 nm resolution reveals that the 150 kDa subunits form a superstructure composed of two segmented and twisted strands. Each strand is 12.5 nm in width and composed of 11 segments that enclose a central channel.     

CD8(+), alphabeta-TCR(+), and gammadelta-TCR(+) cells in the recipient hematopoietic environment mediate resistance to engraftment of allogeneic donor bone marrow

Historically, conditioning for engraftment of hematopoietic stem cells has been nonspecific. In the present study, we characterized which cells in the recipient hematopoietic microenvironment prevent allogeneic marrow engraftment. Mice defective in production of alphabeta-TCR(+), gammadelta-TCR(+), alphabeta- plus gammadelta-TCR(+), CD8(+), or CD4(+) cells were transplanted with MHC-disparate allogeneic bone marrow. Conditioning with 500 cGy total body irradiation (TBI) plus a single dose of cyclophosphamide (CyP) on day +2 establishes chimerism in normal recipients. When mice were conditioned with 300 cGy TBI plus a single dose of CyP on day +2, all engrafted, except wild-type controls and those defective in production of CD4(+) T cells. Mice lacking both alphabeta- and gammadelta-TCR(+) cells engrafted without conditioning, suggesting that both alphabeta- and gammadelta-TCR T cells in the host play critical and nonredundant roles in preventing engraftment of allogeneic bone marrow. CD8 knockout (KO) mice engrafted without TBI, but only if they received CyP on day +2 relative to the marrow infusion, showing that a CD8(-) cell was targeted by the CyP conditioning. The CD8(+) cell effector function is mechanistically different from that for conventional T cells, and independent of CD4(+) T helper cells because CD4 KO mice require substantially higher levels of conditioning than the other KO phenotypes. These results suggest that a number of cell populations with different mechanisms of action mediate resistance to engraftment of allogeneic marrow. Targeting of specific recipient cellular populations may permit conditioning approaches to allow mixed chimerism with minimal morbidity and could potentially avoid the requirement for myelotoxic agents altogether.