Inhibition of Escherichia coli CTP synthase by glutamate gamma-semialdehyde and the role of the allosteric effector GTP in glutamine hydrolysis. (57/471)

Cytidine 5'-triphosphate synthase catalyses the ATP-dependent formation of CTP from UTP with either ammonia or glutamine as the source of nitrogen. When glutamine is the substrate, GTP is required as an allosteric effector to promote catalysis. Escherichia coli CTP synthase, overexpressed as a hexahistidine-tagged form, was purified to high specific activity with the use of metal-ion-affinity chromatography. Unfused CTP synthase, generated by the enzymic removal of the hexahistidine tag, displayed an activity identical with that of the purified native enzyme and was used to study the effect of GTP on the inhibition of enzymic activity by glutamate gamma-semialdehyde. Glutamate gamma-semialdehyde is expected to inhibit CTP synthase by reacting reversibly with the active-site Cys-379 to form an analogue of a tetrahedral intermediate in glutamine hydrolysis. Indeed, glutamate gamma-semialdehyde is a potent linear mixed-type inhibitor of CTP synthase with respect to glutamine (K(is) 0.16+/-0.03 mM; K(ii) 0.4+/-0.1 mM) and a competitive inhibitor with respect to ammonia (K(i) 0.39+/-0.06 mM) in the presence of GTP at pH 8.0. The mutant enzyme (C379A), which is fully active with ammonia but has no glutamine-dependent activity, is not inhibited by glutamate gamma-semialdehyde. Although glutamate gamma-semialdehyde exists in solution primarily in its cyclic form, Delta(1)-pyrroline-5-carboxylate, the variation of inhibition with pH, and the weak inhibition by cyclic analogues of Delta(1)-pyrroline-5-carboxylate (L-proline, L-2-pyrrolidone and pyrrole-2-carboxylate) confirm that the rare open-chain aldehyde species causes the inhibition. When ammonia is employed as the substrate in the absence of GTP, the enzyme's affinity for glutamate gamma-semialdehyde is decreased approx. 10-fold, indicating that the allosteric effector, GTP, functions by stabilizing the protein conformation that binds the tetrahedral intermediate(s) formed during glutamine hydrolysis.  (+info)

The role of calcium in the activation of glycogen phosphorylase in the fat body of the fruit beetle, Pachnoda sinuata, by hypertrehalosaemic hormone. (58/471)

The role of calcium in the mediation of the hypertrehalosaemic signal of the endogenous neuropeptide Mem-CC was investigated in vitro and in vivo in the cetoniid beetle Pachnoda sinuata. The presence of Mem-CC increases the influx of extracellular 45Ca(2+) into the fat body as well as the efflux of 45Ca(2+) from pre-loaded fat body into the incubation medium. Extracellular calcium is essential to exert maximal activation of the fat body glycogen phosphorylase by saturating doses of Mem-CC (0.3 nM). This effect of extracellular Ca(2+) is dose-dependent: maximal activation of glycogen phosphorylase by Mem-CC is achieved at calcium concentrations of approximately 1.2 mM and the ED(50) was calculated to be 0.6 mM. Both, thimerosal and thapsigargin caused a stimulation of carbohydrate metabolism in the fat body, suggesting that a release of calcium from the endoplasmic reticulum is involved in this process. However, neither entry of extracellular calcium nor the release from the endoplasmic reticulum are sufficient alone for a full activation of the phosphorylase. The results of the present study suggest that calcium from extracellular as well as from intracellular sources is part of the second messenger system for the transduction of the hypertrehalosaemic signal of Mem-CC in the fat body of P. sinuata.  (+info)

Modulators with convergent cellular actions elicit distinct circuit outputs. (59/471)

Six neuromodulators [proctolin, Cancer borealis tachykinin-related peptide Ia, crustacean cardioactive peptide (CCAP), red pigment-concentrating hormone, TNRNFLRFamide, and pilocarpine] converge onto the same voltage-dependent inward current in stomatogastric ganglion (STG) neurons of the crab C. borealis. We show here that each of these modulators acts on a distinct subset of pyloric network neurons in the STG. To ask whether the differences in cell targets could account for their differential effects on the pyloric rhythm, we systematically compared the motor patterns produced by proctolin and CCAP. The motor patterns produced in proctolin and CCAP differed quantitatively in a number of ways. Proctolin and CCAP both act on the lateral pyloric neuron and the inferior cardiac neuron. Proctolin additionally acts on the pyloric dilator (PD) neurons, the pyloric (PY) neurons, and the ventricular dilator neuron. Using the dynamic clamp, we introduced an artificial peptide-elicited current into the PD and PY neurons, in the presence of CCAP, and converted the CCAP rhythm into a rhythm that was statistically similar to that seen in proctolin. This suggests that the differences in the network effects of these two modulators can primarily be attributed to the known differential distributions of their receptors onto distinct subsets of neurons, despite the fact that they activate the same current.  (+info)

Priming effects of substance P on calcium changes evoked by interleukin-8 in human neutrophils. (60/471)

The neurokinin (NK) substance P (SP), which is a mediator of neurogenic inflammation, has been reported to prime human polymorphonuclear neutrophils (PMNs). The priming effects of SP on PMNs activated by recombinant interleukin-8 (rIL-8) were investigated. SP enhanced, in a dose- and time-dependent way, the rise in cytosolic free-calcium concentration, [Ca(2+)]i, evoked by the chemokine. The priming effects of SP were abolished by exposing PMNs to a calcium-free medium supplemented with EGTA. The C-terminal peptides SP(4-11) and SP(6-11) but not the N-terminal peptide SP(1-7) shared the priming effects of SP. The selective NK-1 receptor agonist [Sar-9, MetO2-11]SP mimicked the effects of SP, which were not reproduced by the selective NK-2 receptor agonist [betaAla-8]-NKA(4-10) or the selective NK-3 agonist senktide. Two selective NK-1 antagonists, CP96,345 and L703,606, dose dependently inhibited SP priming effects. These results demonstrated that SP primes PMNs exposed to rIL-8 and suggested that SP priming effects are receptor mediated.  (+info)

Effects of substance P on identified neurons of the rat dorsal motor nucleus of the vagus. (61/471)

Previous evidence suggests that substance P (SP) activates subpopulations of neurons within the dorsal motor nucleus of the vagus (DMV). In this study we aimed at identifying these subpopulations in relation to their gastrointestinal projection organs or vagal branches and characterizing pharmacologically the SP response. Using whole cell patch-clamp recordings from identified gastrointestinal-projecting vagal motoneurons, we found that SP induced an inward current in all neuronal groups except for cecum-projecting cells. The lowest percentage of SP-responding neurons was found in fundus-projecting cells, where SP also had a concentration-response curve that was shifted to the left (P < 0.05). Independently from the projections, the SP response was reduced by sendide and MEN 10,376 and mimicked by a combination of [Sar(9)-Met(O(2))(11)]SP and alpha-neurokinin. SP and alpha-neurokinin also increased the frequency, but not the amplitude, of postsynaptic currents. In conclusion, we demonstrated that SP induces both pre- and postsynaptic effects on DMV neurons via activation of neurokinin NK(1) and NK(2) receptors. The magnitude of the SP response was correlated to the peripheral target organ.  (+info)

Effect of the hemoregulatory peptide (pEEDCK)2 (pyroGlu-Glu-Asp-Cys-Lys)2 and MIP-1alpha is reduced in bone marrow cultures from patients with chronic myeloid leukemia (CML). (62/471)

The granulocyte-derived hemoregulatory peptide pyroGlu-Glu-Asp-Cys-Lys = pEEDCK is known to keep hematopoietic cells quiescent. When oxidized to its dimeric form (pEEDCK)2, it activates growth of hematopoietic progenitors in association with stroma-derived cytokines. (pEEDCK)2 has a Cys-Cys motif which is also a typical feature of the macrophage inflammatory protein (MIP-1alpha). The present study was designed to analyze differences between the response of normal and leukemic progenitor cells to (pEEDCK)2 or MIP-1alpha. When long-term bone marrow cultures (LTBMCs) were incubated with (pEEDCK)2 or MIP-1alpha and/or cytokines, the stimulatory effect on colony-forming units-granulocyte/erythroid/macrophage/megakaryocyte of LTBMC from chronic myeloid leukemia (CML) patients was less than 50% compared to LTBMC from healthy humans. No difference in oncogene expression could be observed in LTBMC from CML patients regarding reduction of Philadelphia chromosome-associated transcription of the BCR-ABL gene. With respect to the expression of growth and differentiation-associated genes (Galpha16, 5-lipoxygenase, phospholipaseA2, c-kit, and CD34), which were analyzed from LTBMC by semiquantitative reverse transcriptase-polymerase chain reaction, the same transcription rate was observed in CML patients and healthy donors. However, two isoforms of a key enzyme of oxidative metabolism, carnitine palmitoyltransferase (CPT1A and CPT1B), showed 50-fold higher expression rates in LTBMC cells of healthy donors compared to CML patients. It is known that a decrease in oxidative metabolism is associated with an increase in redox equivalents in malignancy. This might result in a reduction of disulphide bonds in (pEEDCK)2 or MIP-1alpha, thus inducing a downregulation of these factors in bone marrow from CML patients.  (+info)

L-pyroglutamate spontaneously formed from L-glutamate inhibits growth of the hyperthermophilic archaeon Sulfolobus solfataricus. (63/471)

Identification of physiological and environmental factors that limit efficient growth of hyperthermophiles is important for practical application of these organisms to the production of useful enzymes or metabolites. During fed-batch cultivation of Sulfolobus solfataricus in medium containing L-glutamate, we observed formation of L-pyroglutamic acid (PGA). PGA formed spontaneously from L-glutamate under culture conditions (78 degrees C and pH 3.0), and the PGA formation rate was much higher at an acidic or alkaline pH than at neutral pH. It was also found that PGA is a potent inhibitor of S. solfataricus growth. The cell growth rate was reduced by one-half by the presence of 5.1 mM PGA, and no growth was observed in the presence of 15.5 mM PGA. On the other hand, the inhibitory effect of PGA on cell growth was alleviated by addition of L-glutamate or L-aspartate to the medium. PGA was also produced from the L-glutamate in yeast extract; the PGA content increased to 8.5% (wt/wt) after 80 h of incubation of a yeast extract solution at 78 degrees C and pH 3.0. In medium supplemented with yeast extract, cell growth was optimal in the presence of 3.0 g of yeast extract per liter, and higher yeast extract concentrations resulted in reduced cell yields. The extents of cell growth inhibition at yeast extract concentrations above the optimal concentration were correlated with the PGA concentration in the culture broth. Although other structural analogues of L-glutamate, such as L-methionine sulfoxide, glutaric acid, succinic acid, and L-glutamic acid gamma-methyl ester, also inhibited the growth of S. solfataricus, the greatest cell growth inhibition was observed with PGA. We also observed that unlike other glutamate analogues, N-acetyl-L-glutamate enhanced the growth of S. solfataricus. This compound was stable under cell culture conditions, and replacement of L-glutamate with N-acetyl-L-glutamate in the medium resulted in increased cell density.  (+info)

Antidiabetic effect of a prodrug of cysteine, L-2-oxothiazolidine-4-carboxylic acid, through CD38 dimerization and internalization. (64/471)

CD38 is a bifunctional enzyme synthesizing (ADP-ribosyl cyclase) and degrading (cyclic ADP-ribose (cADPR) hydrolase) cADPR, a potent Ca(2+) mobilizer from intracellular pools. CD38 internalization has been proposed as a mechanism by which the ectoenzyme produced intracellular cADPR, and thiol compounds have been shown to induce the internalization of CD38. Here, we show that the disulfide bond between Cys-119 and Cys-201 in CD38 may be involved in CD38 dimerization and internalization. We tested the effect of a reducing agent, l-2-oxothiazolidine-4-carboxylic acid (OTC), a prodrug of cysteine, on CD38 internalization in pancreatic islets. OTC enhanced insulin release from isolated islets as well as CD38 internalization and cytoplasmic Ca(2+) level. Furthermore, islet cells treated with antisense CD38 oligonucleotide showed inhibition of OTC-induced insulin secretion. Intake of OTC in db/db mice ameliorated glucose tolerance, insulin secretion, and morphology of islets when compared with control mice. These data indicate that OTC improves glucose tolerance by enhancing insulin secretion via CD38/cADPR/Ca(2+) signaling machinery. Thus, OTC may represent a novel class of antidiabetic drug.  (+info)