Statistical decoding of potent pools based on chemical structure.

Pooling experiments are used as a cost-effective approach for screening chemical compounds as part of the drug discovery process in pharmaceutical companies. When a biologically potent pool is found, the goal is to decode the pool, i.e., to determine which of the individual compounds are potent. We propose augmenting the data on pooled testing with information on the chemical structure of compounds in order to complete the decoding process. This proposal is based on the well-known relationship between biological potency of a compound and its chemical structure. Application to real data from a drug discovery process at GlaxoSmithKline reveals a 100% increase in hit rate, namely, the number of potent compounds identified divided by the number of tests required.

Identification of the Fc epsilonRI-activated tyrosine kinases Lyn, Syk, and Zap-70 in human basophils.

BACKGROUND: In human blood basophils, cross-linking the high-affinity IgE receptor Fc epsilonRI with multivalent antigen activates a signaling pathway leading to Ca2+ mobilization, actin polymerization, shape changes, secretion, and cytokine production. METHODS AND RESULTS: The role of tyrosine kinases in human Fc epsilonRI signaling was explored by using human basophils isolated by Percoll gradient centrifugation followed by negative and/or positive selection with antibody-coated magnetic beads. Fc epsilonRI cross-linking of more than 95% pure basophil preparations activates the protein-tyrosine kinases Lyn and Syk, previously linked to Fc epsilonRI-coupled rodent mast cell activation, as well as Zap-70, previously implicated in T-cell receptor signaling, and causes the tyrosine phosphorylation of multiple proteins. The presence of Lyn, Syk, and Zap-70 in basophils was confirmed by Western blotting in lysates of highly purified basophils and independently by confocal fluorescence microscopy in cells labeled simultaneously with kinase-specific antibodies and with the basophil-specific antibody 2D7. Comparable amounts of Lyn and Syk were found in basophils and B cells, whereas T cells appear to have greater amounts of Zap-70 than basophils. The tyrosine kinase inhibitor piceatannol spares IgE-mediated Lyn activation but inhibits IgE-induced Syk and Zap-70 activation as well as overall protein tyrosine phosphorylation and secretion. Overall protein-tyrosine phosphorylation increases steadily over a range of anti-IgE concentrations that are low to optimal for secretion. However, tyrosine phosphorylation continues to increase at high anti-IgE concentrations that elicit very little secretion (the characteristic high-dose inhibition of secretion). CONCLUSIONS: Our data demonstrate the association of anti-IgE-stimulated, protein-tyrosine phosphorylation by a cascade of tyrosine kinases, including Zap-70 as well as Lyn and Syk, with the initiation of Fc epsilonRI-mediated signaling in human basophils.

Distinct functions of the Fc epsilon R1 gamma and beta subunits in the control of Fc epsilon R1-mediated tyrosine kinase activation and signaling responses in RBL-2H3 mast cells.

In RBL-2H3 rat tumor mast cells, cross-linking the high affinity IgE receptor, Fc epsilon R1, activates the protein-tyrosine kinases Lyn and Syk and initiates a series of responses including protein-tyrosine phosphorylation, inositol 1,4,5-trisphosphate synthesis, Ca2+ mobilization, secretion, membrane ruffling, and actin plaque assembly. The development of chimeric receptors containing cytoplasmic domains of individual subunits of the heterotrimeric (alpha beta gamma 2) Fc epsilon R1 has simplified analyses of early signaling events in RBL-2H3 cells. Here, RBL-2H3 cells were transfected with cDNAs encoding the extracellular and transmembrane domains of the interleukin-2 receptor alpha subunit (the Tac antigen) joined to the C-terminal cytoplasmic domains of the Fc epsilon R1 gamma and beta subunits (TT gamma and TT beta). Both sequences contain tyrosine activation motifs implicated in antigen receptor signal transduction. TT gamma and TT beta are expressed independently of the native Fc epsilon R1, as demonstrated by the ability of Tac cross-linking agents to trigger the clustering and internalization through coated pits of both chimeric receptors without co-clustering the Fc epsilon R1. A full range of signaling activities is induced by TT gamma cross-linking; the TT gamma-induced responses are slower and, except for Lyn activation, smaller than the Fc epsilon R1-induced responses. In striking contrast, TT beta cross-linking elicits no tyrosine phosphorylation or signaling responses, it impairs basal activities measured in secretion and anti-PY (anti-phosphotyrosine antibody) immune complex kinase assays, and it antagonizes Fc epsilon R1-induced Lyn and Syk activation, protein-tyrosine phosphorylation, and signaling responses. We hypothesize that the isolated beta subunit binds a specific kinase or coupling protein(s) required for signaling activity, sequestering it from the signal-transducing gamma subunit. Binding the same kinase or coupling protein to the beta subunit of the intact Fc epsilon R1 may serve instead to present it to the adjacent gamma subunit, resulting in enhanced kinase activation and signaling responses.

Impaired secretion and increased insolubilization of IgE-receptor complexes in mycophenolic acid-treated (guanine nucleotide-depleted) RBL-2H3 mast cells.

In RBL-2H3 rat leukemic mast cells, cross-linking anti-DNP IgE-receptor complexes with multivalent antigen (DNP-BSA) activates a signal transduction pathway leading to Ca2+ influx and secretion. Cross-linking IgE-receptor complexes also stimulates a pathway that inactivates (desensitizes) receptors; this pathway becomes important at high concentrations of cross-linking antigen. Recent evidence that antigen-induced secretion is impaired by mycophenolic acid (MPA), an inhibitor of guanine nucleotide synthesis de novo, has implicated a GTP-binding protein (G protein) in the signaling pathway. Other recent studies have indicated that the conversion of cross-linked receptors to a detergent-insoluble (cytoskeleton-associated) form at high antigen concentrations is correlated with the loss of signaling activity. Here we show that secretion elicited by an optimal concentration of antigen (0.05 micrograms/ml DNP-BSA) is only inhibited by about 25% in guanine nucleotide-depleted cells, whereas secretion elicited by 5 micrograms/ml DNP-BSA, a concentration in the range that causes the high-dose inhibition of secretion, is inhibited by more than 60%. We also show that IgE-receptor complexes are insolubilized in response to 5 but not 0.05 micrograms/ml DNP-BSA in both control and guanine nucleotide-depleted cells. Importantly, the extent of insolubilization elicited by 5 micrograms/ml DNP-BSA is increased by more than 60% in the guanine nucleotide-depleted samples. These results raise the possibility that guanine nucleotide depletion reduces the secretory response to high antigen concentrations in two ways: by inhibiting the G protein-coupled signaling pathway and by increasing the availability of receptors to the pathway leading to receptor insolubilization and inactivation.

Late cases of toxic oil syndrome: evidence that the aetiological agent persisted in oil stored for up to one year.

The symptoms of toxic oil syndrome (TOS), an epidemic that occurred in central and north-western Spain, developed in the great majority of patients during May or June 1981. We now describe the clinical and epidemiological data of five patients with TOS whose onset of symptoms was considerably later than the great majority of cases. In June 1982, one person became symptomatic as a result of consuming a suspect oil two months earlier. Four members of a family that started consuming a suspect oil in November 1981 became ill in December 1981. These data indicate that the aetiological agent of TOS persisted in stored oil for periods as long as one year. The apparent stability of the TOS aetiological agent increases the likelihood of its continued presence in significant concentrations in oils that have been stored since 1981. Thus, the use of such oils in further in vivo and in vitro toxicological studies may yet lead to the isolation and identification of the causal agent of TOS.

The association of oil ingestion with toxic oil syndrome in two convents.

The authors studied the pattern of occurrence of toxic oil syndrome, a previously undescribed disease that occurred in Spain in epidemic form in 1981, in two convents in Madrid. In one convent, the disease affected 66% of 35 novices and nuns who ingested oil from a suspect source, but none of 56 laywomen who ate the same meals but used a different type of oil. In the second convent, in which nuns were also exposed but laywomen were not, 98% of 43 nuns developed toxic oil syndrome compared with none of 70 laywomen. These findings support the hypothesis that a food oil transmitted the etiologic agent of toxic oil syndrome.

The ionic basis of chemotaxis. Separate cation requirements for neutrophil orientation and locomotion in a gradient of chemotactic peptide.

The behavior of cells undergoing chemotaxis may be analyzed in terms of their orientation, a static characteristic, and of their locomotion. We have examined the extracellular divalent cation requirements for orientation and locomotion of rabbit polymorphonuclear leukocytes (neutrophils) in a gradient of the chemotactic peptide N-formyl-methionyl-leu-cyl-phenylalanine (F-Met-Leu-Phe) using the chemotaxis chamber recently developed by Zigmond. This chamber allows direct observation of cells attached to glass coverslips as they move up a gradient of chemotactic agent established across a 1-mm bridge. The orientation of neutrophils in the direction of the gradient was equally efficient whether cells and F-Met-Leu-Phe were suspended in merium supplemented with both Ca2+ and Mg2+ (complete medium), with Mg2+ but not Ca2+ (by simple omission of Ca2+ or by addition of EGTA), or with nonsupplemented medium (by omission of Ca2+ and Mg2+ or by addition of EDTA). These data confirm and extend Zigmond's earlier observation that exogenous divalent cations are not required for polymorphonuclear leukocyte orientation toward the chemotactic peptide. In contrast, cell locomotion, determined by linking the chemotaxis chamber to a time-lapse videocassette recorder and TV monitor, is markedly affected by the medium's content of divalent cations. Cells suspended in medium supplemented with Mg2+ but not calcium (by omission or chelation) or in nonsupplemented medium moved on the average 25% more rapidly than cells in complete Ca2+ and Mg2+ medium. Although the simple omission of Mg2+ does not prevent chemotaxis, chelation of Mg2+ in the medium completely abolishes leukocyte locomotion. Addition of varying concentrations of Mg2+ to the buffer in the presence of EDTA established that cell movement is fully restored by Mg2+ concentrations in the range of 3 X 10(-9) M, concentrations easily attained in the absence of added Mg2+. It was concluded that neither Ca2+ nor Mg2+ is needed for orientation in response to F-Met-Leu-Phe. However, low levels of exogenous Mg2+ but not Ca2+ are required for effective locomotion of neutrophils in the Zigmond changer. This result contrasts with data obtained in the Boyden chamber, where exogenous Ca2+ is considered essential for maximum chemotactic response.

A possible role for 5-phosphoribosyl 1-pyrophosphate in the stimulation of uterine purine nucleotide synthesis in response to oestradiol-17 .

1. It has been reported that the rate of purine nucleotide synthesis de novo in the immature rat uterus is doubled at 6h after administration of oestradiol-17beta. The present work confirms an increased incorporation of glycine and adenine into uterine nucleotides between 2 and 6h after hormone treatment and investigates the mechanism of this response. 2. Activation of regulatory enzymes is unlikely to promote increased nucleotide synthesis: the activities of 5-phosphoribosyl 1-pyrophosphate amidotransferase (EC 2.4.2.14) and adenine phosphoribosyltransferase (EC 2.4.2.7) are the same in uterine extracts from control and oestrogen-treated rats. 3. Therefore it was proposed that oestradiol might promote an increased supply of a rate-limiting substrate. The low oestrogen-sensitive rate of AMP synthesis from adenine and endogenous 5-phosphoribosyl 1-pyrophosphate in the intact uterus compared with the high, oestrogen-insensitive rate in uterine extracts supplemented with 5-phosphoribosyl 1-pyrophosphate is evidence that the supply of 5-phosphoribosyl 1-pyrophosphate limits purine nucleotide formation and may increase after hormone treatment. This proposal is supported by the decrease in AMP synthesis in the whole tissue in the presence of guanine and 7-amino-3-(beta-d-ribofuranosyl)pyrazolo[3,4-d]pyrimidine (formycin). These compounds do not inhibit adenine uptake or adenine phosphoribosyltransferase activity, but they both decrease the availability of 5-phosphoribosyl 1-pyrophosphate, the former by promoting its utilization by hypoxanthine/guanine phosphoribosyltransferase (EC 2.4.2.8) and the latter by inhibiting its synthesis from ribose 5-phosphate and ATP by ribose 5-phosphate pyrophosphokinase (EC 2.7.6.1). 4. It is unlikely that the increased availability of 5-phosphoribosyl 1-pyrophosphate results from hormonal stimulation of ribose 5-phosphate formation. Methylene Blue and phenazine methosulphate both increase ribose 5-phosphate without altering the supply of 5-phosphoribosyl 1-pyrophosphate. 5. The activity of ribose 5-phosphate pyrophosphokinase is low in uterine extracts and increases rapidly in response to oestradiol. Therefore the hormonal activation of the routes of purine nucleotide synthesis both de novo and from preformed precursors may be due, at least in part, to an increased availability of the common rate-limiting substrate 5-phosphoribosyl 1-pyrophosphate, mediated by activation of ribose 5-phosphate pyrophosphokinase.