Microarray data quality analysis: lessons from the AFGC project

Genome-wide expression profiling with DNA microarrays has and will provide a great deal of data to the plant scientific community. However, reliability concerns have required the development data quality tests for common systematic biases. Fortunately, most large-scale systematic biases are detectable and some are correctable by normalization. Technical replication experiments and statistical surveys indicate that these biases vary widely in severity and appearance. As a result, no single normalization or correction method currently available is able to address all the issues. However, careful sequence selection, array design, experimental design and experimental annotation can substantially improve the quality and biological of microarray data. In this review, we discuss these issues with reference to examples from the Arabidopsis Functional Genomics Consortium (AFGC) microarray project.     

Iron supplementation moderates but does not cure the Belgrade anemia

Belgrade rats inherit microcytic, hypochromic anemia as an autosomalrecessive trait (gene symbol b). Erythrocytes and tissue are iron deficientin the face of elevated TIBC (total iron binding capacity) and percent ironsaturation; iron injections increased the number of erythrocytes but theirappearance remained abnormal. We have investigated iron supplements toimprove husbandry of b/b rats and to learn more about the underlying defectand its tissue distribution. Weekly IM (intramuscular) injections ofiron–dextran (Imferon at 30 mg kg) improved the anemia but did not alter thered cell morphology. Certain diets also improved the health of b/b rats whencompared to standard rat chows by the criteria of weight, survival toadulthood, hematology and reproduction. The critical nutritional factorturned out to be iron bioavailability, with ferrous iron added to the dietimproving the health of Belgrade rats without affecting the underlyingerythroid defect. Tissue iron measurements after dietary or parenteralsupplementation confirmed the iron deficient status of untreated b/b rats andestablished that dietary ferrous iron partially relieved this deficiency,with injections leading to greater amounts of tissue iron. Serum iron andTIBC were also found to be elevated in untreated b/b rats, with dietarysupplementation decreasing but not eliminating the elevation in TIBC. Thesestudies indicate that iron supplements can improve the health of b/b ratswithout altering the underlying defect and also suggest that the mutationcould alter iron uptake in the GI (gastrointestinal) tract.     

Detection of expiratory flow limitation during exercise in COPD patients

Koulouris, Nickolaos G., Ioanna Dimopoulou, PäiviValta, Richard Finkelstein, Manuel G. Cosio, and J. Milic-Emili.Detection of expiratory flow limitation during exercise in COPDpatients. J. Appl. Physiol. 82(3):723-731, 1997.The negative expiratory pressure (NEP) method wasused to detect expiratory flow limitation at rest and at differentexercise levels in 4 normal subjects and 14 patients with chronicobstructive pulmonary disease (COPD). This method does not requireperformance of forced expirations, nor does it require use of bodyplethysmography. It consists in applying negative pressure (5cmH₂O) at the mouth during early expiration and comparing the flow-volume curve of the ensuing expiration with that of the preceding control breath. Subjects in whomapplication of NEP does not elicit an increase in flow during part orall of the tidal expiration are considered flow limited. The fournormal subjects were not flow limited up to 90% of maximal exercisepower output(_max).Five COPD patients were flow limited at rest, 9 were flow limited atone-third _max, and 12 were flow limited at two-thirds_max. Whereasin all patients who were flow limited at rest the maximalO₂ uptake was below the normallimits, this was not the case in most of the other patients. Inconclusion, NEP provides a rapid and reliable method to detectexpiratory flow limitation at rest and during exercise.

     

The water permeability of toad urinary bladder. II. The value of Pf/Pd(w) for the antidiuretic hormone-induced water permeation pathway

Using the methods described in the preceding paper (Levine et al., 1984) for measuring the magnitude of the water-permeable barriers in series with the luminal membrane, we correct measured values of Pd(w) in bladders stimulated with low doses of antidiuretic hormone (ADH) or 8-bromo cyclic AMP to obtain their true values in the luminal membrane. Simultaneously, we also determine Pf. We thus are able to calculate Pf/Pd(w) for the hormone-induced water permeation pathway in the luminal membrane. Our finding is that Pf/Pd(w) approximately equal to 17. Two channel models consistent both with this value and the impermeability of the ADH-induced water permeation pathway to small nonelectrolytes are: (a) a long (approximately equal to 50 A), small- radius (approximately equal to 2 A) pore through which 17 water molecules pass in single-file array, and (b) a shower-head-like structure in which the stem is long and of large radius (approximately equal to 20 A) and the cap has numerous short, small-radius (approximately equal to 2 A) pores. A third possibility is that whereas the selective permeability to H2O results from small-radius (approximately equal to 2 A) pores, the large value of Pf/Pd(w) arises from their location in the walls of long tubular vesicles (approximately 2 micron in length and 0.1 micron in diameter) that are functionally part of the luminal membrane after having fused with it. Aggregate-containing tubular vesicles of these dimensions have been reported to fuse with the luminal membrane in response to ADH stimulation and have been implicated in the ADH-induced hydroosmotic response.     

Preferential oxidative dehalogenation upon conversion of 2-halophenols by Rhodococcus opacus 1G

The regiospecificity of hydroxylation of C2-halogenated phenols by Rhodococcus opacus 1G was investigated. Oxidative defluorination at the C2 position ortho with respect to the hydroxyl moiety was preferred over hydroxylation at the non-fluorinated C6 position for all 2-fluorophenol compounds studied. Initial hydroxylation of 2,3, 5-trichlorophenol resulted in the exclusive formation of 3, 5-dichlorocatechol. These results indicate that, in contrast to all other phenol ortho-hydroxylases studied so far, phenol hydroxylase from R. opacus 1G is capable of catalyzing preferential oxidative defluorination but also oxidative dechlorination.     

Dysregulation of T Lymphocyte Proliferative Responses in Autoimmunity

T cells are critically dependent on cellular proliferation in order to carry out their effector functions. Autoimmune strains are commonly thought to have uncontrolled T cell proliferation; however, in the murine model of autoimmune diabetes, hypo-proliferation of T cells leading to defective AICD was previously uncovered. We now determine whether lupus prone murine strains are similarly hyporesponsive. Upon extensive characterization of T lymphocyte activation, we have observed a common feature of CD4 T cell activation shared among three autoimmune strains–NOD, MRL, and NZBxNZW F1s. When stimulated with a polyclonal mitogen, CD4 T cells demonstrate arrested cell division and diminished dose responsiveness as compared to the non-autoimmune strain C57BL/6, a phenotype we further traced to a reliance on B cell mediated costimulation, which underscores the success of B cell directed immune therapies in preventing T cell mediated tissue injury. In turn, the diminished proliferative capacity of these CD4 T cells lead to a decreased, but activation appropriate, susceptibility to activation induced cell death. A similar decrement in stimulation response was observed in the CD8 compartment of NOD mice; NOD CD8 T cells were distinguished from lupus prone strains by a diminished dose-responsiveness to anti-CD3 mediated stimulation. This distinction may explain the differential pathogenetic pathways activated in diabetes and lupus prone murine strains.     

Steady-state kinetics of the hypoxanthine phosphoribosyltransferase from Trypanosoma cruzi

The kinetic mechanism for the reaction catalyzed by the hypoxanthine phosphoribosyltransferase (HPRT) from Trypanosoma cruzi was analyzed to determine the feasibility of designing a parasite-specific mechanism-based inhibitor of this enzyme. The results show that the HPRT from T. cruzi follows an essentially ordered bi–bi reaction, and like its human counterpart also likely forms a dead end complex with purine substrates and the product pyrophosphate. Computational fitting of the kinetics data to multiple initial velocity equations gave results that are consistent with the dead end complex arising when the hypoxanthine- or guanine-bound form of the enzyme binds pyrophosphate rather than the phosphoribosylpyrophosphate substrate of the productive forward reaction. Limited proteolytic digestion was employed to provide additional support for formation of the dead end complex and to estimate the K_d values for substrates of both the forward and reverse reactions. Due to similarities with the kinetic mechanism of the human HPRT, the results reported here for the HPRT from T. cruzi indicate that the design of a mechanism-based inhibitor of the trypanosomal HPRT, that would not also inhibit the human enzyme, may be difficult. However, the results also show that a potent selective inhibitor of the trypanosomal HPRT might be achieved via the design of a bi-substrate type inhibitor that incorporates analogs of moieties for a purine base and pyrophosphate.     

Ion and nonelectrolyte permeability properties of channels formed in planar lipid bilayer membranes by the cytolytic toxin from the sea anemone,Stoichactis helianthus

Summary When present at nanomolar concentrations on one side of a lipid bilayer membrane,helianthus toxin (a protein of mol wt16,000) increases enormously membrane permeability to ions and nonelectrolytes by forming channels in the membrane. Membranes containing sphingomyelin are especially sensitive to toxin, but sphingomyelin isnot required for toxin action. Conductance is proportional to about the 4th power of toxin concentration. Single channel conductances are approximately 2×10^–10 mho in 0.1m KCl. Toxin-treated membranes are more permeable to K⁺ and Na⁺ than to Cl^– and SO ₄ ⁼ , but the degree of selectivity is pH dependent. Above pH 7 membranes are almost ideally selective for K⁺ with respect to SO ₄ ⁼ , whereas below pH 4 they are poorly selective. The channels show classical molecular sieving for urea, glycerol, glucose, and sucrose — implying a channel radius >5 Å. In symmetrical salt solutions above pH 7, theI–V characteristic of the channel shows significant rectification: below pH 5 there is very little rectfication. Because of the effects of pH on ion selectivity and channel conductance, and also because of the rectification in symmetrical salt solutions and the effect of pH on this, we conclude that there are titratable negative charge groups in the channel modulating ion permeability and selectivity. Since pH changes on the side containing the toxin are effective whereas pH changes on the opposite side are not, we place these negative charges near the mouth of the channel facing the solution to which toxin was added.