Isotopic hydrogen exchange in reactions catalysed by cysteine lyase and serine sulphhydrase.

Serine sulphhydrase from chicken liver and cysteine lyase from chicken-embryo yolk sac catalyse the exchange of alpha-H atoms of the amino acid substrate with 3-H-2O. The degree of labelling of the unreacted substrate approaches a maximum of one atom per mol of amino acid. In the absence of replacing agent there is practically no H-exchange in the substrate. The alpha-H of the accumulating beta-substitution product is completely replaced by the labelled hydrogen of the solvent water, irrespective of the duration of incubation. The amount of labelled alpha-hydrogen incorporated into excess (unreacted) amino acids substrate within 3.5-h incubation is somewhat less than the amount incorporated into the product of the complete enzymic beta-replacement reaction. Within the sensitivity limits of detection, the enzymes do not induce any isotopic exchange either of b-H atoms in the amino substrate or of 18-O-labelled beta-HO groups, in the case of L-serine. Neither serine sulphhydrase nor cysteine lyase will catalyse alpha-hydrogen exchange in close structural analogues of their substrates, e.g. L-alanine, D-serine, threonin, 3-phosphoserine. A special case is the interaction of cysteine lyase with the competitive inhibitor, L-serine (whose inhibitor constant, K-i, is equal to the Michaelis constant, K-m, of L-cysteine): the lyase catalyses, only in presence of a cosubstrate thiol, alpha-H exchange in L-serine at approximately the same rate as in L-cysteine. The present data concerning isotopic alpha-H exchange in substrate amino acids, and evidence published earlier, suggest that the catalytic mechanism of replacement-specific beta-lyases may significantly differ from that of the eliminating or ambivalent (mixed-function) lyases. Formation of alpha, beta-unsaturated pyridoxylidene aldimines as real reaction intermediates is unlikely in the case of lyases specifically catalysing beta-replacement reactions; these may proceed by some alternative mechanism of the type suggested in this paper.     

Trypanosoma cruzi: inhibition by spirogermanium hydrochloride

Spirogermanium is an antineoplastic agent that has been shown to be useful for the treatment of a variety of solid tumors and Plasmodium falciparum infection. We found that this agent, at concentrations of 1-10 micrograms/ml, markedly inhibited the growth of epimastigotes of Trypanosoma cruzi. This inhibition of growth was seen in liver infusion tryptose cultures as well as on agar where colonial growth was inhibited markedly. Ultrastructural studies demonstrated that affected organisms were round and swollen and contained vacuoles, lamellar structures, and multivesicular bodies. Spirogermanium also significantly decreased the growth of intracellular amastigotes in myotubes. Pretreatment of myotubes with the agent protected them from infection with trypomastigotes but tachyzoites of Toxoplasma sp. readily infected pretreated cells. These data suggest that spirogermanium may be useful as a chemotherapeutic agent against T. cruzi.     

Perturbation Biology: Inferring Signaling Networks in Cellular Systems

We present a powerful experimental-computational technology for inferring network models that predict the response of cells to perturbations, and that may be useful in the design of combinatorial therapy against cancer. The experiments are systematic series of perturbations of cancer cell lines by targeted drugs, singly or in combination. The response to perturbation is quantified in terms of relative changes in the measured levels of proteins, phospho-proteins and cellular phenotypes such as viability. Computational network models are derived de novo, i.e., without prior knowledge of signaling pathways, and are based on simple non-linear differential equations. The prohibitively large solution space of all possible network models is explored efficiently using a probabilistic algorithm, Belief Propagation (BP), which is three orders of magnitude faster than standard Monte Carlo methods. Explicit executable models are derived for a set of perturbation experiments in SKMEL-133 melanoma cell lines, which are resistant to the therapeutically important inhibitor of RAF kinase. The resulting network models reproduce and extend known pathway biology. They empower potential discoveries of new molecular interactions and predict efficacious novel drug perturbations, such as the inhibition of PLK1, which is verified experimentally. This technology is suitable for application to larger systems in diverse areas of molecular biology.     

Functional Modulation of the Glutamate Transporter Variant GLT1b by the PDZ Domain Protein PICK1

The dominant glutamate transporter isoform in the mammalian brain, GLT1, exists as at least three splice variants, GLT1a, GLT1b, and GLT1c. GLT1b interacts with the scaffold protein PICK1 (protein interacting with kinase C1), which is implicated in glutamatergic neurotransmission via its regulatory effect on trafficking of AMPA-type glutamate receptors. The 11 extreme C-terminal residues specific for the GLT1b variant are essential for its specific interaction with the PICK1 PDZ domain, but a functional consequence of this interaction has remained unresolved. To identify a functional effect of PICK1 on GLT1a or GLT1b separately, we employed the Xenopus laevis expression system. GLT1a and GLT1b displayed similar electrophysiological properties and EC₅₀ for glutamate. Co-expressed PICK1 localized efficiently to the plasma membrane and resulted in a 5-fold enhancement of the leak current in GLT1b-expressing oocytes with only a minor effect on [³H]glutamate uptake. Three different GLT1 substrates all caused a slow TBOA-sensitive decay in the membrane current upon prolonged application, which provides support for the leak current being mediated by GLT1b itself. Leak and glutamate-evoked currents in GLT1a-expressing oocytes were unaffected by PICK1 co-expression. PKC activation down-regulated GLT1a and GLT1b activity to a similar extent, which was not affected by co-expression of PICK1. In conclusion, PICK1 may not only affect glutamatergic neurotransmission by its regulatory effect on glutamate receptors but may also affect neuronal excitability via an increased GLT1b-mediated leak current. This may be particularly relevant in pathological conditions such as amyotrophic lateral sclerosis and cerebral hypoxia, which are associated with neuronal GLT1b up-regulation.