Residues glutamate 216 and aspartate 301 are key determinants of substrate specificity and product regioselectivity in cytochrome P450 2D6

Cytochrome P450 2D6 (CYP2D6) metabolizes a wide range of therapeutic drugs. CYP2D6 substrates typically contain a basic nitrogen atom, and the active-site residue Asp-301 has been implicated in substrate recognition through electrostatic interactions. Our recent computational models point to a predominantly structural role for Asp-301 in loop positioning (Kirton, S. B., Kemp, C. A., Tomkinson, N. P., St.-Gallay, S., and Sutcliffe, M. J. (2002) Proteins 49, 216-231) and suggest a second acidic residue, Glu-216, as a key determinant in the binding of basic substrates. We have evaluated the role of Glu-216 in substrate recognition, along with Asp-301, by site-directed mutagenesis. Reversal of the Glu-216 charge to Lys or substitution with neutral residues (Gln, Phe, or Leu) greatly decreased the affinity (K(m) values increased 10-100-fold) for the classical basic nitrogen-containing substrates bufuralol and dextromethorphan. Altered binding was also manifested in significant differences in regiospecificity with respect to dextromethorphan, producing enzymes with no preference for N-demethylation versus O-demethylation (E216K and E216F). Neutralization of Asp-301 to Gln and Asn had similarly profound effects on substrate binding and regioselectivity. Intriguingly, removal of the negative charge from either 216 or 301 produced enzymes (E216A, E216K, and D301Q) with elevated levels (50-75-fold) of catalytic activity toward diclofenac, a carboxylate-containing CYP2C9 substrate that lacks a basic nitrogen atom. Activity was increased still further (>1000-fold) upon neutralization of both residues (E216Q/D301Q). The kinetic parameters for diclofenac (K(m) 108 microm, k(cat) 5 min(-1)) along with nifedipine (K(m) 28 microm, k(cat) 2 min(-1)) and tolbutamide (K(m) 315 microm, k(cat) 1 min(-1)), which are not normally substrates for CYP2D6, were within an order of magnitude of those observed with CYP3A4 or CYP2C9. Neutralizing both Glu-216 and Asp-301 thus effectively alters substrate recognition illustrating the central role of the negative charges provided by both residues in defining the specificity of CYP2D6 toward substrates containing a basic nitrogen.     

Insertional mutagenesis of the arginine cage domain of the gonadotropin-releasing hormone receptor

The pattern of side-chain conservation at the cytoplasmic side of the third transmembrane domain of rhodopsin family G protein-coupled receptors, Asp/Glu-Arg-Tyr/X-X-X-Ile/Val, defines a structural "arginine cage" domain. Previous computational and mutagenesis studies of the GnRH receptor indicated an important contribution of local interactions to the function of this domain. We have investigated the functional importance of the intrahelical position and orientation of the arginine cage using insertional mutagenesis. Introduction of a single Ala proximal to the conserved Asp-Arg of this domain caused loss of detectable ligand binding. Inserting a second Ala, however, restored high-affinity agonist binding. Further insertion of three or four Ala residues at this site generated receptors that bound agonist with an affinity 3- to 10-fold higher than that of the wild-type receptor. Loss of detectable coupling to inositol phosphate turnover in all these mutant receptors confirms that the structure required in this region for efficient signaling is highly constrained. In contrast, the recovery of agonist binding with the progressive insertion of two to four Ala residues indicates that specific orientations of this segment can stabilize high-affinity receptor conformations that are uncoupled from signal transduction.     

Axonal protein synthesis provides a mechanism for localized regulation at an intermediate target

As axons grow past intermediate targets, they change their responsiveness to guidance cues. Local upregulation of receptor expression is involved, but the mechanisms for this are not clear. Here protein synthesis is traced within individual axons by introducing RNAs encoding visualizable reporters. Individual severed axons and growth cones can translate proteins and also export them to the cell surface. As axons reach the spinal cord midline, EphA2 is among the receptors upregulated on at least some distal axon segments. Midline reporter upregulation is recapitulated by part of the EphA2 mRNA 3' untranslated region, which is highly conserved and includes known translational control sequences. These results show axons contain all the machinery for protein translation and cell surface expression, and they reveal a potentially general and flexible RNA-based mechanism for regulation localized within a subregion of the axon.     

Atractis marquezi n. sp. (Nematoda: Atractidae) and a revision of Atractis dujardin, 1845, sensu Baker, 1987

Atractis marquezi n. sp. from the large intestine of the tortoise Geochelone nigrita is described and illustrated. Atractis Dujardin, 1845, sensu Baker, 1987, is revised to contain only those atractids having a lagonoid spicule. The remaining species of Atractis sensu Baker, 1987, are assigned to Cyrtosomum, Pseudatractis, and Klossinemella. A. marquezi represents the 12th species to be assigned to this genus and is distinguished from other Neotropical species by the distribution pattern of caudal papillae of the male: 2 pairs precloacal, 2 pairs adcloacal, and 6 pairs postcloacal.     

Domain-specific olivocerebellar projection regulated by the EphA-ephrin-A interaction

Neural maps in the vertebrate central nervous system often show discontinuously segregated, domain-to-domain patterns. However, the molecular mechanism that establishes such maps is not well understood. Here we show that in the chicken olivocerebellar system, EphA receptors and ephrin-As are expressed with distinct levels and combinations in mapping domains. When ephrin-A2 is retrovirally overexpressed in the cerebellum, the olivocerebellar map is disrupted, excluding axons with high receptor activity from ectopic expression domains. Conversely, overexpression of a truncated EphA3 receptor in the cerebellum reduces endogenous ligand activity to undetectable levels and causes aberrant mapping, with high receptor axons invading high ligand domains. In vitro, ephrin-A2 inhibits outgrowth of inferior olive axons in a region-specific manner. These results suggest that Eph receptors and ephrins constitute domain-specific positional information, and the spatially accurate receptor-ligand interaction is essential to guide inferior olive axons to their correct target domains.     

Genetic analysis of ephrin-A2 and ephrin-A5 shows their requirement in multiple aspects of retinocollicular mapping

Ephrin-A2 and -A5 are thought to be anteroposterior mapping labels for the retinotectal/retinocollicular projection. Here, gene disruptions of both these ephrins are characterized. Focal retinal labeling reveals moderate map abnormalities when either gene is disrupted. Double heterozygotes also have a phenotype, showing an influence of absolute levels. In vitro assays indicate these ephrins are required for repellent activity in the target and also normal responsiveness in the retina. In double homozygotes, anteroposterior order is almost though not completely lost. Temporal or nasal retinal labelings reveal quantitatively similar but opposite shifts, with multiple terminations scattered widely over the target. These results indicate an axon competition mechanism for mapping, with a critical role for ephrins as anteroposterior topographic labels. Dorsoventral topography is also impaired, showing these ephrins are required in mapping both axes.     

Reversible G(1) arrest induced by inhibition of the epidermal growth factor receptor tyrosine kinase requires up-regulation of p27(KIP1) independent of MAPK activity

We have used quinazoline inhibitors of the epidermal growth factor receptor (EGFR) tyrosine kinase to study the link between EGFR signaling and G(1) to S traverse. Treatment of A431 and MDA-468 human tumor cells with 0.1-10 microM AG-1478 inhibited basal and ligand-stimulated EGFR phosphorylation without a decrease in receptor content, EGF-binding sites, or binding affinity. Incubation of A431 cells with 0.1-1 microM AG-1517 abrogated (125)I-EGF internalization. Both AG-1478 and AG-1517 markedly inhibited A431 and MDA-468 colony formation in soft agarose at concentrations between 0.01 and 1 microM. Daily injections of AG-1478 at 50 mg/kg delayed A431 tumor formation in athymic nude mice. A transient exposure of A431 cells to AG-1478 resulted in a dose-dependent up-regulation of the cyclin-dependent kinase inhibitor p27, down-regulation of cyclin D1 and of active MAPK, and hypophosphorylation of the retinoblastoma protein (Rb). These changes were temporally associated with recruitment of tumor cells in G(1) phase and a marked reduction of the proportion of cells in S phase. Upon removal of the kinase inhibitor, EGFR and Rb phosphorylation and the levels of cyclin D1 protein were quickly restored, but the cells did not reenter S phase until p27 protein levels were decreased. Phosphorothioate p27 oligonucleotides decreased p27 protein in A431 cells and abrogated the quinazoline-mediated G(1) arrest. Treatment of A431 cells with PD 098509, a synthetic inhibitor of MEK1, inhibited MAPK activity without inducing G(1) arrest or increasing the levels of p27. However, treatment with LY 294002, an inhibitor of phosphatidylinositol 3-kinase (PI3K), inhibited basal Akt activity, up-regulated p27, and recruited cells in G(1). These data suggest that p27 is required for the growth arrest that follows interruption of the EGFR kinase in receptor-overexpressing cells. In addition, the G(1) arrest and up-regulation of p27 resulting from EGFR blockade are not due to the interruption of MAPK, but to the interruption of constitutively active PI3K function.     

Genomic structure and expression of parathyroid hormone-related protein gene (PTHrP) in a teleost, Fugu rubripes

In this study we describe the isolation and characterisation of the parathyroid hormone-related protein (PTHrP) gene from the teleost Fugu rubripes. The gene has a relatively simple structure, compared with tetrapod PTHrP genes, composed of three exons and two introns, encompassing 2.25 kb of genomic DNA. The gene encodes a protein of 163 amino acids, with a putative signal peptide of 37 amino acids and a mature peptide of 126 amino acids. The overall homology with known tetrapod PTHrP proteins is low (36%), with a novel sequence inserted between positions 38 and 65, the absence of the conserved pentapeptide (TRSAW) and shortened C-terminal domain. The N-terminus shows greater conservation (62%), suggesting that it may have a hypercalcaemic function similar to that of tetrapod PTHrP. In situ localisation and RT–PCR have demonstrated the presence of PTHrP in a wide range of tissues with varying levels of expression. Sequence scanning of overlapping cosmids has identified three additional genes, TMPO, LDHB and KCNA1, which map to human chromosome 12, with the latter two mapping to 12p12-11.2. PTHrP in human also maps to this chromosome 12 sub-region, thus demonstrating conservation of synteny between human and Fugu.     

Biodegradation of Trichloroethylene and Dichloromethane in Contaminated Soil and Groundwater

Soil column and serum bottle microcosm experiments were conducted to investigate the potential for in situ anaerobic bioremediation of trichloroethy lene (TCE) and dichloromethane (DCM) at the Pinellas site near Largo, Florida. Soil columns with continuous groundwater recycle were used to evaluate treatment with complex nutrients (casamino acids, methanol, lactate, sulfate); benzoate and sulfate; and methanol. The complex nutrients drove microbial dechlorination of TCE to ethene, whereas the benzoate/sulfate and methanol supported microbial dechlorination of TCE only to cis-1 ,2-dichloroethylene (cDCE). Microbial sulfate depletion in the benzoate/sulfate column allowed further dechlorination of cDCE to vinyl chloride. Serum bottle microcosms were used to investigate TCE dechlorination and DCM biodegradation in Pinellas soil slurries bioaugmented with liquid from the soil columns possessing TCE-dechlorinating activity and DCM biodegradation by indigenous microorganisms. Bioaugmented soil microcosms showed immediate TCE dechlorination in the microcosms with methanol or complex nutrients, but no dechlorination in the benzoate/sulfate microcosm. DCM biodegradation by indigenous microorganisms occurred in soil microcosms amended with either benzoate/sulfate or methanol, but not with complex nutrients. Bioaugmentation stimulated DCM biodegradation in both complex nutrient and methanol-amended microcosms, but appeared to inhibit DCM biodegradation in benzoate/sulfate-amended microcosms. TCE dechlorination occurred before DCM biodegradation in bioaugmented microcosms when both compounds were present.