Modulation of striatal enkephalinergic neurons by antipsychotic drugs

In this paper we review the detailed mechanisms underlying the modulation of enkephalinergic neurons by dopaminergic neurons in rat striatum. Several lines of evidence, which showed that striatal levels of [Met5]enkephalin (ME) increase after the nigrostriatal dopaminergic pathway was interrupted by hemitransection or direct administration of 6-hydroxydopamine to the substantia nigra, or after repeated injections of either reserpine or haloperidol, suggest that dopamine (DA) plays an important role in regulating the metabolism of ME-containing neurons in the striatum. The increase in ME content after repeated injections of haloperidol was found in areas heavily innervated by DA neurons such as striatum or nucleus accumbens but not in hypothalamus, brain stem, and hippocampus. Further studies suggest that striatal cholinergic interneurons may partially mediate the action of haloperidol on enkephalinergic neurons. Several studies have been carried out to determine whether the elevation of striatal ME content after haloperidol treatment was caused by an increase in the synthesis or by a decrease in the utilization of ME. The rate of decline of striatal ME content in haloperidol-treated rats was steeper than that of controls after intraventricular injection of cycloheximide, which indicated that haloperidol accelerates the turnover of ME. This hypothesis was confirmed by our recent findings that the level of mRNA coding for preproenkephalin A, determined by cell-free translation and blot hybridization with cDNA clones, is increased after repeated injections of haloperidol.     

风胁迫对三种叶菜的机械损伤及预测模型

为了研究风害对不同叶菜的影响,本研究通过模拟风洞试验,以上海青,四季小白菜,玻璃生菜3种叶菜为试验材料,分别在5,15,25 m/s风速条件下设置5,10,15 min的风胁迫处理,采用电导率法、伤口染色目测法、伤口色泽L值测定法研究风胁迫对不同种类叶菜造成的机械损伤,并对以上3种测定方法进行了综合评价,进而建立相应的数学模型。结果表明:风速和风胁迫时间两因子均对3种叶菜的相对电导率、目测等级、L值有显著性影响;两者的交互作用对上海青和四季小白菜的相对电导率有显著性影响,但对玻璃生菜的相对电导率无显著影响;另外,两者的交互作用对3种叶菜的目测等级均影响显著,但对3种叶菜的L值影响均不显著。25 m/s和15 m/s风对3种叶菜都可引起显著机械损伤,其中,在25 m/s持续15 min风处理下机械损伤最为严重,在此处理组合下,上海青、四季小白菜和玻璃生菜的相对电导率分别高于对照214.70%,228.96%,266.92%;目测等级分别高于对照2.3,2.4和3.6级;L值分别低于对照21.17%,38.91%,42.73%。显然,与上海青和四季小白菜相比,玻璃生菜更容易受到风害影响。Gauss2D拟合模型中,3种叶菜机械损伤拟合模型的决定系数R2均超过0.95,证明该拟合模型能较好地预测不同叶菜遭受风害后的机械损伤程度,可以为风害机械损伤预测提供理论基础。     

Canine reduction in early man: A critique of three mechanical models

The possibility that projecting maxillary canines interfere with either a «rotary chewing» form of molar occlusion or the lateral excursion of the mandible has been used to suggest two dietary (non-weapon) selection models for canine reduction in the earliest male humans. A third model explaining canine reduction is based on the idea that a projecting mandibular canine could interfere with its tip-to-tip occlusion with the maxillary lateral incisor. In this paper, these three mechanical models are critically reexamined in light of more recent studies of occlusion in extant primates, detailed observations of anterior tooth morphology and wear in Miocene to Recent anthropoids, cheek tooth microwear inA. afarensis, and the currently accepted phylogeny and fossil record of the great apes and man.     

Depression of potassium-evoked striatal acetylcholine release by delta-receptor activation: inhibition by cholinoactive agents

To examine the role of delta-opioid receptors in the modulation of striatal acetylcholine (ACh) release, the action of D-Pen2,L-Pen5-enkephalin, a selective delta-opioid receptor agonist, was tested on [3H]ACh release from slices of the rat caudate-putamen. Slices, incubated with [3H]choline, were superfused with a physiological buffer and stimulated twice by exposure to a high potassium (K+) concentration. In the absence of a cholinesterase inhibitor, 1 microM D-Pen2,L-Pen5-enkephalin produced a 46 and 35% decrease in the release of [3H]ACh evoked by 15 and 25 mM K+, respectively. The depressant action of the enkephalin analogue was concentration dependent, with a maximal effect on K+-evoked [3H]ACh release occurring at 1.0 microM, and was completely blocked in the presence of the delta-opioid receptor selective antagonist, ICI 174864 (1 microM). In the presence of the cholinesterase inhibitors physostigmine (10 microM) and neostigmine (10 microM), or the muscarinic receptor agonist oxotremorine (10 microM), D-Pen2,L-Pen5-enkephalin did not depress the K+-evoked release of [3H]ACh. Atropine (1 microM) blocked the inhibitory effect of physostigmine on the depressant action of D-Pen2,L-Pen5-enkephalin. The results of this study indicate that delta-opioid receptor activation is associated with an inhibition of striatal ACh release, but this opioid-cholinergic interaction is not apparent under conditions of presynaptic muscarinic receptor activation.     

Modulation of inhibition of ferrochelatase by N-methylprotoporphyrin

Protoporhyrin IX ferrochelatase catalyses the terminal step of the haem-biosynthetic pathway by inserting ferrous iron into protoporphyrin IX. NMPP (N-methylprotoporphyrin), a transition-state analogue and potent inhibitor of ferrochelatase, is commonly used to induce haem deficiency in mammalian cell cultures. To create ferrochelatase variants with different extents of tolerance towards NMPP and to understand further the mechanism of ferrochelatase inhibition by NMPP, we isolated variants with increased NMPP resistance, bearing mutations in an active-site loop (murine ferrochelatase residues 248-257), which was previously shown to mediate a protein conformational change triggered by porphyrin binding. The kinetic mechanisms of inhibition of two variants, in which Pro255 was replaced with either arginine (P255R) or glycine (P255G), were investigated and compared with that of wild-type ferrochelatase. While the binding affinity of the P255X variants for NMPP decreased by one order of magnitude in relation to that of wild-type enzyme, the inhibition constant increased by approximately two orders of magnitude (K(i)(app) values of 1 microM and 2.3 microM for P255R and P255G respectively, as against 3 nM for wild-type ferrochelatase). Nonetheless, the drastically reduced inhibition of the variants by NMPP was not paralleled with a decrease in specificity constant (kcat/K(m, protoporhyrin IX)) and/or catalytic activity (kcat). Further, although NMPP binding to either wild-type ferrochelatase or P255R occurred via a similar two-step kinetic mechanism, the forward and reverse rate constants associated with the second and rate-limiting step were comparable for the two enzymes. Collectively, these results suggest that Pro255 has a crucial role in maintaining an appropriate protein conformation and modulating the selectivity and/or regiospecificity of ferrochelatase.     

Modulation of striatal dopamine release in cerebral ischemia byL-arginine

The effect ofL-arginine, the precursor of nitric oxide, on ischemic dopamine release from the striatum was investigated in Mongolian gerbils subjected to bilateral carotid artery occlusion (15 min) alone or with reflow (2 h). Dopamine and its metabolites were measured in the striatal extracellular space dialysate after continuous perfusion (2 l/min) of artificial extracellular fluid in the presence or absence of 15 mmol/literL- orD-arginine or 1 mmol/liter nitro-L-arginine.L-Arginine but notD-arginine increased the striatal content of dopamine in pre- and postischemia whereas it lowered the levels of dopamine and 3-methoxytyramine induced by ischemia. In contrast, nitro-L-arginine reduced the preischemic levels of dopamine and 3,4-dihydroxyphenyl-acetic acid, and had no effect on the ischemic release of dopamine. These findings indicate thatL-arginine stereospecifically modified the ischemic release and metabolism of dopamine. The data also suggest that the basal level of nitric oxide is not involved in dopamine release during ischemia but may participate in regulating dopamine release under physiological conditions.Presented in part at the 19th International Joint Conference on Stroke and Cerebral Circulation, San Diego, California, February 17–19, 1994.     

Modulation of oxidative damage by nitroxide free radicals

Piperidine nitroxides like 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) are persistent free radicals in non-acidic aqueous solutions and organic solvents that may have value as therapeutic agents in medicine. In biological environments, they undergo mostly reduction to stable hydroxylamines but can also undergo oxidation to reactive oxoammonium compounds. Reactions of the oxoammonium derivatives could have adverse consequences including chemical modification of vital macromolecules and deleterious effects on cell signaling. An examination of their reactivity in aqueous solution has shown that oxoammonium compounds can oxidize almost any organic as well as many inorganic molecules found in biological systems. Many of these reactions appear to be one-electron transfers that reduce the oxoammonium to the corresponding nitroxide species, in contrast to a prevalence of two-electron reductions of oxoammonium in organic solvents. Amino acids, alcohols, aldehydes, phospholipids, hydrogen peroxide, other nitroxides, hydroxylamines, phenols and certain transition metal ions and their complexes are among reductants of oxoammonium, causing conversion of this species to the paramagnetic nitroxide. On the other hand, thiols and oxoammonium yield products that cannot be detected by ESR even under conditions that would oxidize hydroxylamines to nitroxides. These products may include hindered secondary amines, sulfoxamides and sulfonamides. Thiol oxidation products other than disulfides cannot be restored to thiols by common enzymatic reduction pathways. Such products may also play a role in cell signaling events related to oxidative stress. Adverse consequences of the reactions of oxoammonium compounds may partially offset the putative beneficial effects of nitroxides in some therapeutic settings.     

Reversible inhibition of cathepsin L-like proteases by 4-mer pseudopeptides.

A library of 121 pseudopeptides was designed to develop reversible inhibitors of trypanosomal enzymes (cruzain from Trypanosoma cruzi and congopain from Trypanosoma congolense). The peptides share the framework: Cha-X1-X2-Pro (Cha=cyclohexyl-alanine, X1 and X2 were phenylalanyl analogs), based on a previous report [Lecaille, F., Authié, E., Moreau, T., Serveau, C., Gauthier, F. and Lalmanach, G. (2001) Eur. J. Biochem. 268, 2733-2741]. Five peptides containing a nitro-substituted aromatic residue (Tyr/Phe) and one a 4-chloro-phenylalanine at the X1 position, and 3-(2-naphthyl)-alanine, homocyclohexylalanine or 3-nitro-tyrosine (3-NO(2)-Tyr) at the X2 position, were selected. They inhibited congopain more effectively than cruzain, except Cha-4-NO(2)-Phe-3-NO(2)-Tyr-Pro which bound the two parasitic enzymes similarly. Among this series, Cha-3-NO(2)-Tyr-HoCha-Pro and Cha-4-NO(2)-Phe-3-NO(2)-Tyr-Pro are the most selective for congopain relative to host cathepsins. No hydrolysis occurred upon prolonged incubation time with purified enzymes. In addition introduction of non-proteogenic residues in the peptidyl backbone greatly enhanced resistance to proteolysis by mammalian sera.     

Hemoglobin-degrading, aspartic proteases of blood-feeding parasites: substrate specificity revealed by homology models

Blood-feeding parasites, including schistosomes, hookworms, and malaria parasites, employ aspartic proteases to make initial or early cleavages in ingested host hemoglobin. To better understand the substrate affinity of these aspartic proteases, sequences were aligned with and/or three-dimensional, molecular models were constructed of the cathepsin D-like aspartic proteases of schistosomes and hookworms and of plasmepsins of Plasmodium falciparum and Plasmodium vivax, using the structure of human cathepsin D bound to the inhibitor pepstatin as the template. The catalytic subsites S5 through S4' were determined for the modeled parasite proteases. Subsequently, the crystal structure of mouse renin complexed with the nonapeptidyl inhibitor t-butyl-CO-His-Pro-Phe-His-Leu [CHOHCH(2)]Leu-Tyr-Tyr-Ser- NH(2) (CH-66) was used to build homology models of the hemoglobin-degrading peptidases docked with a series of octapeptide substrates. The modeled octapeptides included representative sites in hemoglobin known to be cleaved by both Schistosoma japonicum cathepsin D and human cathepsin D, as well as sites cleaved by one but not the other of these enzymes. The peptidase-octapeptide substrate models revealed that differences in cleavage sites were generally attributable to the influence of a single amino acid change among the P5 to P4' residues that would either enhance or diminish the enzymatic affinity. The difference in cleavage sites appeared to be more profound than might be expected from sequence differences in the enzymes and hemoglobins. The findings support the notion that selective inhibitors of the hemoglobin-degrading peptidases of blood-feeding parasites at large could be developed as novel anti-parasitic agents.     

Modulation of oxidative events by multivalent manganese complexes in brain tissue

Manganese toxicity can evoke neuropsychiatric and neuromotor symptoms, which have frequently been attributed to profound oxidative stress in the dopaminergic system. However, the characterization of manganese as a pro-oxidant remains controversial because antioxidant properties also have been associated with this metal. The current study was designed to address these disparate findings concerning the oxidative properties of manganese. The apparent ability of manganese in its divalent form to promote formation of reactive oxygen species (ROS) within a cortical mitochondrial-synaptosomal (P2) fraction was completely abolished by the addition of one five hundredth of its molarity of desferroxamine (DFO), a trivalent metal chelator. This large ratio and the high specificity of DFO for trivalent metal ions discounted the possibility of inhibition of ROS generation by direct sequestration of divalent manganese, and implied the trace presence of a trivalent metal. Further analysis suggested that this trace metal was manganic rather than ferric ion. Ferric ion was able to dampen the reactive oxygen species-generating capacity of manganous chloride, whereas manganic ion markedly promoted this property attributed to manganous ion. Such findings of the potent effects of trace amounts of trivalent cations upon Mn2+-related free radical generation offer resolution of earlier disparate findings concerning the oxidative character of manganese.     

Digestion of the lambda cI repressor with various serine proteases and correlation with its three dimensional structure

Partial proteolysis of the lambda cI repressor has been carried out systematically with trypsin, chymotrypsin, elastase, endoproteinase Glu-C, kallikrein, and thrombin. The cleavage sites have been determined by (i) comparison of fragments produced and observed in SDS-polyacrylamide gel with known fragments and plots of distance migrated versus log (molecular weight of fragment), (ii) partial Edman sequencing of the stable C-terminal fragments to identify cleavage points, and (iii) electrospray mass spectrometry of fragments produced. Most cleavage points are found to occur in the region 86-137, saving some in the N-terminal domain observed for trypsin and Glu-C. Region 86-137 can be further subdivided into three regions 86-91, 114-121, and 128-137 prone to cleavage, with intermediate regions resistant to cleavage to all six proteases. These resistant regions show that much of the region 93-131 previously called a 'linker' is actually part of the C-domain as first proposed in all models from our laboratory. Region 92-114 includes the cleavage site Ala-Gly, which must be buried in the intact repressor. The observed cleavage points in region 114-137 can be used to judge the best among three previously proposed models since they differ from each other in the structure of region 93-131. Model 1j5g is adjudged to be better than model 1lwq (which is based on 1kca, a crystal structure) as susceptible residues are more exposed in the former and lack of cleavages at six sites is better explained. Likewise, the models 1j5g and 1lwq are compared with a recent crystal structure of fragment 101-229 in 2ho0 and another low resolution crystal structure in 3bdn.     

Irreversible inhibition of serine proteases by peptidyl derivatives of alpha-aminoalkylphosphonate diphenyl esters

Peptidyl alpha-aminoalkylphosphonate diphenyl esters have been synthesized and shown to be effective inhibitors of serine proteases. Extending the peptide chain from a single alpha-aminoalkylphosphonate residue (kobs/[I] = 2.5-260 M-1 s-1) to a tripeptide or tetrapeptide derivative (kobs/[I] = 7,000-17,000 M-1 s-1) resulted in 65-2800 improvement in inhibitory potency and increased specificity. The rate of inactivation of chymotrypsin by MeO-Suc-Ala-Ala-Pro-HNCH(CH2Ph)P(O)(OPh)2 was decreased 5 fold in the presence of the substrate Suc-Val-Pro-Phe-NA (0.119 mM). Phosphonylated serine proteases are extremely stable since the half-life for reactivation was greater than 48 hrs for the inhibited elastases and at least 10 hrs for chymotrypsin.