Rescue of abnormal phenotypes of the delta2 glutamate receptor-null mice by mutant delta2 transgenes

The delta2 glutamate receptor (GluRdelta2) has a crucial role in cerebellar functions; disruption of GluRdelta2 alleles in mice (delta2(-/-)) impairs synapse formation and long-term depression, which is thought to underlie motor learning in the cerebellum, and consequently leads to motor discoordination. However, it has been unclear whether GluRdelta2 is activated by glutamate analogues. Here we introduced a GluRdelta2 transgene, which had a mutation (Arg514Lys) in the putative ligand-binding motif conserved in all mammalian ionotropic glutamate receptors (iGluRs) and their ancestral bacterial periplasmic amino-acid-binding proteins, into delta2(-/-) mice. Surprisingly, a mutant GluRdelta2 transgene, as well as a wild-type GluRdelta2 transgene, rescued all abnormal phenotypes of delta2(-/-) mice. Therefore, these results indicate that the conserved arginine residue, which is crucial for the binding of iGluRs to glutamate analogues, is not essential for the restoration of GluRdelta2 functions in delta2(-/-) mice.     

Isomerization of delta 1-piperideine-2-carboxylate to delta 2-piperideine-2-carboxylate on complexation with flavoprotein D-amino acid oxidase.

The 1,646 cm-1 band in a resonance Raman spectrum obtained with excitation in the charge-transfer band of the complex of oxidized D-amino acid oxidase (DAO) with the oxidation product of D-lysine catalyzed by DAO shifted to 1,617 cm-1 upon 2-13C substitution of lysine. Thus, the band is assigned to a C(2) = C(3) stretching mode of the enamine, delta 2-piperideine-2-carboxylate (En). In the enzyme-free solution, the product is preferentially in the cyclic imine form, delta 1-piperideine-2-carboxylate (Im). Thus, DAO has a higher affinity for the enamine form than for the imine form. The pH effects on the affinity of DAO for the product and on the molar absorption coefficient at 630 nm in the charge-transfer band, suggest that the enzyme-bound product is En in the neutral form at the N atom. As the value of observed rate constant between DAO and the product was constant at high product concentrations, the binding mechanism can be explained as follows; E + Im in equilibrium with EIm in equilibrium with EEN: rapid bimolecular and slow unimolecular processes. The isomerization of the imine form to the enamine form proceeds in the slow process. The low affinity of Im for DAO may be due to a steric repulsion of the hydrogen atoms of Im at C(3) in the active site. The hydrogen atoms of a substrate D-amino acid at C(3), which correspond to the C(3) hydrogens of Im, may act repulsively in the active site and the repulsive energy may induce strain or distortion of the substrate and the enzyme, accelerating the catalytic reaction.     

Synthesis of isotopically labeled delta2-isopentenylpyrophosphate

The high yield chemical conversion of acetate to Δ²-isopentenylpyrophosphate is described. This procedure, plus the availability of high specific activity isotopic labels in acetate, allows the efficient synthesis of large quantities of highly labeled Δ²-isopentenylpyrophosphate.     

Occurrence of a long-chain delta 3,delta 2-enoyl-CoA isomerase in rat liver.

Isoproteins of delta 3,delta 2-enoyl-CoA isomerase (EC 5.3.3.8), an auxiliary enzyme in the beta-oxidation of unsaturated fatty acids having double bonds at odd-numbered positions, were studied in livers of control and clofibrate-treated rats. When liver extracts were applied to a hydroxyapatite column at pH 7.0, the previously characterized peroxisomal trifunctional hydratase-dehydrogenase-isomerase enzyme and the mitochondrial isomerase, which shows a preference for short-chain substrates, were eluted almost in parallel. In addition to these activities, a separate isomerase was observed to elute at a lower potassium phosphate concentration in the gradient. Experiments with extracts of purified mitochondria and peroxisomes demonstrated the mitochondrial origin of this third activity. Studies on the kinetic properties of the third isomerase showed that it has a preference for C10-C12 substrates. An Mr of 200,000 was obtained for the native protein by gel-filtration chromatography. Antibodies to mitochondrial short-chain isomerase and peroxisomal trifunctional enzyme did not recognize this novel mitochondrial isoenzyme. The immunological non-cross-reactivity can be interpreted as suggesting that the different isomerases are not closely related at the level of the primary structure of the polypeptide chain. The present data demonstrate that, similar to many other enzymes of beta-oxidation, delta 3,delta 2-enoyl-CoA isomerase has at least three isoenzymes in rat liver: mitochondrial short- and long-chain isomerases and an additional peroxisomal isoenzyme, which in this case is a part of a multifunctional protein.     

Repeated delta1-opioid receptor stimulation reduces delta2-opioid receptor responses in the SA node

Ultra-low-dose methionine-enkephalin-arginine-phenylalanine improves vagal transmission (vagotonic) and decreases heart rate via delta(1)-opioid receptors within the sinoatrial (SA) node. Higher doses activate delta(2)-opioid receptors, interrupt vagal transmission (vagolytic), and reduce the bradycardia. Preconditioning-like occlusion of the nodal artery produced a vagotonic response that was reversed by the delta(1)-antagonist 7-benzylidenaltrexone (BNTX). The following study tested the hypothesis that extended delta(1)-opioid receptor stimulation reduces subsequent delta(2)-receptor responses. The delta(2)-agonist deltorphin II was introduced in the SA node by microdialysis to evaluate delta(2) responses before and after infusion of the delta(1)-agonist TAN-67. TAN-67 reduced the vagolytic effect of deltorphin by two-thirds. When the delta(1)-antagonist BNTX was combined with TAN-67, the deltorphin response was preserved, suggesting that attrition of the prior response was mediated by delta(1) activity. When TAN-67 was omitted in time control studies, some loss of delta(2) responses was apparent in the absence of the delta(1) treatment. This loss was also eliminated by BNTX, suggesting that the attenuation of the response after deltorphin alone was also the result of delta(1) activity. Additional studies tested TAN-67 alone in the absence of prior deltorphin. When time controls were conducted without the initial deltorphin treatment, a robust vagolytic response was observed. When TAN-67 preceded the delayed deltorphin, the vagolytic response was eroded, indicating an independent effect of TAN-67. BNTX infused afterward was unable to restore the delta(2) response. These data support the conclusion that the loss of the delta(2) response resulted from reduced delta(2) activity mediated by continued delta(1)-receptor stimulation and not the arithmetic consequence of increased competition from that same delta(1) receptor.     

Structural studies of duck delta 1 and delta 2 crystallin suggest conformational changes occur during catalysis

Duck delta1 and delta2 crystallin are 94% identical in amino acid sequence, and while delta2 crystallin is the duck orthologue of argininosuccinate lyase (ASL) and catalyzes the reversible breakdown of argininosuccinate to arginine and fumarate, the delta1 isoform is enzymatically inactive. The crystal structures of wild type duck delta1 and delta2 crystallin have been solved at 2.2 and 2.3 A resolution, respectively, and the refinement of the turkey delta1 crystallin has been completed. These structures have been compared with two mutant duck delta2 crystallin structures. Conformational changes were observed in two regions of the N-terminal domain with intraspecies differences between the active and inactive isoforms localized to residues 23-32 and both intra- and interspecies differences localized to the loop of residues 74-89. As the residues implicated in the catalytic mechanism of delta2/ASL are all conserved in delta1, the amino acid substitutions in these two regions are hypothesized to be critical for substrate binding. A sulfate anion was found in the active site of duck delta1 crystallin. This anion, which appears to mimic the fumarate moiety of the argininosuccinate substrate, induces a rigid body movement in domain 3 and a conformational change in the loop of residues 280-290, which together would sequester the substrate from the solvent. The duck delta1 crystallin structure suggests that Ser 281, a residue strictly conserved in all members of the superfamily, could be the catalytic acid in the delta2 crystallin/ASL enzymatic mechanism.     

Novel delta2-isoxazolines as group II phospholipase A2 inhibitors

The synthesized imidazolyl substituted delta2-isoxazolines were subjected to Phospholipase A(2) (PLA(2)) enzyme inhibitory activity against snake venom source and their structure-activity relationship with respect to different groups attached to this moiety is reported for the first time. The crystal structure of the compound 2-butyl-5-chloro-3H-imidazolyl-4-carbaldehyde oxime 2, an intermediate for the construction of isoxazolines is reported. These compounds exerted a significant PLA(2) enzyme inhibitory activity against group II PLA(2). The in vivo activity on mice of selected compounds 3bI and 3bIV shows the comparable anti-inflammatory activity with the known standard ursolic acid.     

The mouse gene PDCR encodes a peroxisomal delta(2), delta(4)-dienoyl-CoA reductase.

Here we describe the identification and characterization of a novel mouse gene, PDCR, that encodes a peroxisomal Delta(2), Delta(4)-dienoyl-CoA reductase. The mouse PDCR cDNA contains an 892-base pair open reading frame and is predicted to encode a 292-amino acid protein with a deduced molecular mass of 31,298 Da that terminates in a consensus type-1 peroxisomal targeting signal. Purified recombinant PDCR protein was generated from Escherichia coli and catalyzed the NADPH-dependent reduction of Delta(2)-trans, Delta(4)-trans-decadienoyl-CoA with a specific activity of 20 units/mg. Enzymatic characterization followed by high pressure liquid chromatography analysis of the products revealed that PDCR converted Delta(2)-trans,Delta(4)-trans-decadienoyl-CoA to a Delta(3)-enoyl-CoA but not to a Delta(2)-enoyl-CoA. Kinetic analyses demonstrated that PDCR is active on a broad range of Delta(2), Delta(4)-dienoyl-CoAs. Although the observed substrate preference was to Delta(2)-trans,Delta(4)-trans-decadienoyl-CoA, PDCR was also active on a C(22) substrate with multiple unsaturations, a result consistent with the role of peroxisomes in the oxidation of complex, very long chain, polyunsaturated fatty acids. The presence of a type-1 peroxisomal targeting signal Ala-Lys-Leu-COOH at the C terminus of PDCR suggested that this protein may be peroxisomal. We observed that tagged PDCR was efficiently transported to the peroxisome lumen in normal human fibroblasts but not in cells derived from a Zellweger syndrome patient with a specific defect in peroxisomal matrix protein import. We conclude that this protein resides within the peroxisome matrix and therefore represents the first mammalian peroxisomal Delta(2),Delta(4)-dienoyl-CoA reductase to be characterized at the molecular level.     

Synergistic antiproliferative effect of delta 12-prostaglandin J2 (delta 12-PGJ2) and hyperthermia on human esophageal cancer cell lines

delta 12-PGJ2, one of the cyclopentenone prostaglandins and the ultimate metabolite of prostaglandin D2, has been reported to have potent antiproliferative activity on various tumor cells in vitro and in vivo. In this study, the combined effect of delta 12-PGJ2 and hyperthermia on six established cell lines of human esophageal carcinoma (SGF series) was analyzed by an in vitro assay, and the degree of apoptosis induced by this combination was examined to clarify the mechanism of supra-additive effects. In five SGF cell lines, except SGF-7 cells, combination therapy with delta 12-PGJ2 and hyperthermia showed synergistic antiproliferative effects. The supra-additive combined effect of delta 12-PGJ2 and hyperthermia on esophageal cancer cells is attributed to the synergistic induction of apoptosis. delta 12-PGJ2 induced G1 accumulation and apoptosis was induced by delta 12-PGJ2 from G1 phase. Hyperthermia induced G1 accumulation and apoptosis was induced by hyperthermia during all cell phases. Both augmented G1 arrest followed by G1 phase-selective induction of apoptosis and increased apoptotic induction without cell-cycle specificity are responsible for the synergism of combined treatment with delta 12-PGJ2 and hyperthermia.     

Cycloheximide reduces PGD2 or delta 12-PGJ2 cytotoxicity on NCG cells

To study the precise mechanism of cytotoxic activity of PGD2 or delta 12-PGJ2 (a biologically active metabolite of PGD2), we examined the effect of various compounds on PGD2 or delta 12-PGJ2 cytotoxicity, using a human neuroblastoma cell line (NCG). Cycloheximide (CHM) specifically protected PGD2 cytotoxicity on NCG cells. When delta 12-PGJ2 was tested, CHM exhibited a similar rescue effect. Puromycin, mitomycin C, and alpha-amanitin did not affect PGD2 or delta 12-PGJ2 cytotoxicity. Emetine showed a variable and no consistent rescue effect CHM may have been active at the primary site where PGD2 or delta 12-PGJ2 exerts its cytotoxicity. This is the first report indicating that CHM reduces the cytotoxicity induced by PGD2 or delta 12-PGJ2.     

delta1-piperideine-2-carboxylate reductase of Pseudomonas putida.

Pseudomonas putida metabolizes D-lysine to delta 1-piperideine-2-carboxylate and L-pipecolate. The second step of this catabolic pathway is catalyzed by delta 1-piperideine-2-carboxylate reductase. This enzyme was isolated and purified from cells grown on DL-lysine as substrate. The enzyme was very unstable, resulting in low recovery of activity and low purity after a six-step purification procedure. The enzyme had a pH optimum of 8.0 to 8.3. The Km values for delta 1-piperideine-2-carboxylate and NADPH were 0.23 and 0.13 mM, respectively. NADPH at concentrations above 0.15 mM was inhibitory to the enzyme. Delta 1-pyrroline-5-carboxylate, pyroglutamate, and NADH were poor substrates or coenzyme for delta 1-piperideine-2-carboxylate reductase. The enzyme reaction from delta 1-piperideine-2-carboxylate to L-pipecolate was irreversible. EDTA, sodium pyrophosphate, and dithiothreitol at concentrations of 1 mM protected the enzyme during storage. The enzyme was inhibited almost totally by Zn2+, Mn2+, Hg2+ Co2+, and p-chloromercuribenzoate at concentrations of 0.1 mM. The enzyme had a molecular weight of about 200,000. Both D-lysine and L-lysine were good inducers for the enzyme. Neither delta1-piperideine-2-carboxylate nor L-pipecolate was an effective inducer for the enzyme. P. putida cells grew on D-lysine only after a 5- to 8-h lag, which could be abolished by adding a supplement of 0.01% alpha-ketoglutarate or other readily metabolizable compounds. Such a supplement also converted the noncoordinate induction of this enzyme and pipecolate oxidase, both of the D-lysine pathway, to coordinacy. However, this effect was not observed if the enzyme pair was from different pathways of lysine metabolism in this organism (i.e., the D- and L-lysine pathways).     

Human V gamma 9-V delta 2 T cell receptor-gamma delta lymphocytes show specificity to Daudi Burkitt's lymphoma cells

Peripheral blood TCR-gamma delta cells with different functional V gamma or V delta gene rearrangements represent two nonoverlapping subsets. The major subset uses the V gamma 9 and the V delta 2 gene segments and the minor subset the V delta 1 gene segments in its functional TCR rearrangement. Upon in vitro activation, these TCR-gamma delta lymphocytes display MHC-unrestricted lytic activity, against a wide variety of tumor cells of distinct histologic origin. Here we show that fresh TCR-gamma delta lymphocytes that express a V gamma 9-V delta 2 encoded TCR display a specific proliferative response to Daudi, Burkitt's lymphoma cells. Moreover, cloned V gamma 9-V delta 2 lymphocytes show the capacity to lyse Daudi cells, whereas none of the cloned V gamma 1 TCR-gamma delta lymphocytes shows such specificity. Nucleotide diversity at the V-D-J junction of the TCR-V delta 2 gene did not contribute to this Daudi cell specificity. Comparison of the MHC-unrestricted cytolytic capacities of the V gamma 9-V delta 2 and the V delta 1 clones using a panel of distinct types of tumor target cells showed that on average, the level of MHC unrestricted lysis of V gamma 9-V delta 2 clones against these tumor cells exceeded that of V delta 1 clones. However, in contrast to all these tumor cell lines, only the Daudi cells showed such an absolute distinction in susceptibility to lysis by V gamma 9-V delta 2 and V delta 1 clones. V gamma 9-V delta 2 clones that were generated with a stimulator cell other than Daudi did not lyse their stimulator cells but nevertheless showed specific cytolysis of Daudi cells. The specific proliferation to and cytolysis of Daudi cells of the entire V gamma 9-V delta 2 subpopulation of TCR-gamma delta lymphocytes is reminiscent of a superantigen response.     

Hb A2-Wrens or alpha 2 delta 2 98(FG5) Val----Met, an unstable delta chain variant identified by sequence analysis of amplified DNA

Data are reported for an 85-year-old black make who had an HPFH condition on one chromosome and a suspected 'delta-thalassemia' on the other. Sequence analysis of amplified DNA of an appropriate segment of the delta-globin gene identified a GTG to ATG mutation for codon 98 and thus a Val----Met replacement in the delta chain. This abnormality was confirmed by hybridization of amplified DNA with 32P-labeled synthetic probes and by the amino-acid composition of the isolated tryptic peptide delta T-11. Thus, the 'delta-thalassemia' is caused by the presence of an Hb A2 variant that is considered to be unstable to a similar extent as is Hb K?ln, its beta chain counterpart.     

Crystallization and preliminary X-ray diffraction studies of mitochondrial short-chain delta 3,delta 2-enoyl-CoA isomerase from rat liver.

Crystals of short-chain delta 3,delta 2-enoyl-CoA isomerase (EC 5.3.3.8) from rat liver mitochondria have been grown using the hanging-drop vapour diffusion technique. The enoyl-CoA isomerase is an auxiliary enzyme in the beta-oxidation pathway of fatty acid metabolism, and catalyzes the isomerization of unsaturated fatty acids to produce the metabolizable delta 2-trans isomer. The crystals belong to the orthorhombic space group P2(1)2(1)2(1) with unit cell dimensions a = 47.9, b = 118.4 and c = 164.8 A, and diffract to 3 A.     

Activation of V gamma 9V delta 2 T cells by NKG2D

Human Vgamma9 Vdelta2 T cells recognize phosphorylated nonpeptide Ags (so called phosphoantigens), certain tumor cells, and cells treated with aminobisphosphonates. NKG2D, an activating receptor for NK cells, has been described as a potent costimulatory receptor in the Ag-specific activation of gammadelta and CD8 T cells. This study provides evidence that Vgamma9 Vdelta2 T cells may also be directly activated by NKG2D. Culture of PBMC with immobilized NKG2D-specific mAb or NKG2D ligand MHC class I related protein A (MICA) induces the up-regulation of CD69 and CD25 in NK and Vgamma9 Vdelta2 but not in CD8 T cells. Furthermore, NKG2D triggers the production of TNF-alpha but not of IFN-gamma, as well as the release of cytolytic granules by Vgamma9 Vdelta2 T cells. Purified Vgamma9 Vdelta2 T cells kill MICA-transfected RMA mouse cells but not control cells. Finally, DAP10, which mediates NKG2D signaling in human NK cells, was detected in resting and activated Vgamma9 Vdelta2 T cells. These remarkable similarities in NKG2D function in NK and Vgamma9 Vdelta2 T cells may open new perspectives for Vgamma9 Vdelta2 T cell-based immunotherapy, e.g., by Ag-independent killing of NKG2D ligand-expressing tumors.     

PKC regulates the delta2 glutamate receptor interaction with S-SCAM/MAGI-2 protein

Inside cells, membrane proteins are localized at particular surface domains to perform their precise functions. Various kinds of PDZ domain proteins have been shown to play important roles in the intracellular trafficking and anchoring of membrane proteins. In this study, we show that delta2 glutamate receptor is interacting with S-SCAM/MAGI-2, a PDZ domain protein localized in the perinuclear region and postsynaptic sites of cerebellar Purkinje cells. The binding is regulated by PKC (protein kinase-C) mediated phosphorylation of the receptor with a unique repetitive structure in S-SCAM/MAGI-2. Co-expression of both proteins resulted in drastic changes of the receptor localization in COS7 cells. These results show a novel regulatory mechanism for the binding of PDZ domain proteins and suggest that the interaction between delta2 receptor and S-SCAM/MAGI-2 may be important for intracellular trafficking of the receptor.     

Formation of thiol conjugates of 9-deoxy-delta 9,delta 12(E)-prostaglandin D2 and delta 12(E)-prostaglandin D2

Albumin catalyzes the transformation of prostaglandin D2 to 9-deoxy-delta 9,delta 12(E)-prostaglandin D2 and to isomeric prostaglandin D2 compounds including delta 12(E)-prostaglandin D2. Both of these compounds are alpha,beta-unsaturated ketones, which should render them susceptible to nucleophilic addition. We therefore examined the ability of the compounds to form conjugates with thiols glutathione and cysteine. During incubation with excess glutathione, both 9-deoxy-delta 9,delta 12(E)-prostaglandin D2 and delta 12(E)-prostaglandin D2 formed a conjugate. Conjugation of 9-deoxy-delta 9,delta 12(E)-prostaglandin D2 occurred very rapidly; approximately 70% was conjugated within 2 min. In contrast, conjugation of delta 12(E)-prostaglandin D2 with glutathione proceeded at a much slower rate; only 38% was conjugated at 60 min. The formation of both conjugates was enhanced by glutathione S-transferase. Conjugation of both compounds with cysteine was found to occur more rapidly than with glutathione. This effect was more pronounced with delta 12(E)-prostaglandin D2 in which 60% conjugated with cysteine within 2 min. These differences are likely attributed to greater steric hindrance for conjugation across the delta 12 double bond compared to that across the delta 9 bond. Analysis by fast atom bombardment mass spectrometry confirmed the formation of the glutathione conjugate of 9-deoxy-delta 9,delta 12(E)-prostaglandin D2. Following prolonged incubation of 9-deoxy-delta 9,delta 12(E)-prostaglandin D2 with excess glutathione in the presence of glutathione S-transferase, a small quantity of a bis conjugate of this compound was also detected by mass spectrometry.(ABSTRACT TRUNCATED AT 250 WORDS)