Alpha-methyl-para-tyrosine pretreatment protects from striatal neuronal death induced by four-vessel occlusion in the rat

Rats were treated with alpha-methyl-para-tyrosine (AMT, 250 mg/kg, i.p), an hydroxylase inhibitor, in order to decrease brain levels of catecholamines. Six hours later, when cerebral dopamine (DA) and norepinephrine were reduced by about 80%, a transient forebrain ischemia of 30 min duration was induced by four-vessel occlusion technique. Evaluation of brain damage 72 hours after ischemia showed that AMT treatment significantly decreased neuronal necrosis in the striatum but had no cytoprotective effect in the CA1 sector of the hippocampus and in the neocortex. AMT treatment reduced mortality within the ischemic period but did not affect either the mortality within the recirculation period or the postischemic neurologic deficit. These results suggest that the striatal cytoprotective effect of AMT is linked to cerebral DA depletion and that excessive release of DA during ischemia or dopaminergic hyperactivity during recirculation play a detrimental role in the development of ischemic cell damage in the striatum.     

Turnover Rate of Brain Acetylcholine Using HPLC Separation of the Transmitter

A simple, reliable method was developed for measuring brain acetylcholine (ACh) turnover using HPLC methodology. Mice were injected intravenously with [3H]choline ([3H]Ch), and the turnover rate of ACh was calculated from the formation of [3H]ACh. Ch and ACh were separated from phosphorylcholine and from other radioactive compounds using tetraphenylboron extraction and counterion/reverse-phase chromatography. Endogenous Ch and ACh were quantified electrochemically through hydrogen peroxide production in a postcolumn reactor containing covalently bonded ACh esterase and Ch oxidase. Labeled Ch and ACh were quantified in the same sample by collecting the chromatographic fractions for radioactive content determinations. The method is rapid, well adapted to large series, and highly reproducible, with recoveries of 72.1% for Ch and 79.3% for ACh. The turnover value in mouse cerebral hemispheres was 16.02 nmol g-1 min-1 and decreased to 9.94 nmol g-1 min-1 in mice treated with oxotremorine.     

Effects of iron-induced lipid peroxidation and of acidosis on choline uptake by synaptosomes

The effects of iron-induced lipid peroxidation and of lactic acidosis on [³H]choline uptake were investigated on crude synaptosomes prepared from rat cerebral cortices. Fe²⁺-induced lipid peroxidation as evidenced from the production of thiobarbituric acid reactives substances (TBARS) was correlated with a decrease in high-affinity choline uptake (HACU). Trolox C, a free radical scavenger, prevented both Fe²⁺-induced TBARS production and decrease in HACU. Lactic acidosis (pH 6.0 for 30 or 60 min) increased the TBARS production with concomitant decrease in HACU (–48%, –78%, respectively). The acidosis dependent decrease was not reversible following pH 7.4 readjustment after 60 min acidosis. It was not prevented by trolox C, although trolox C inhibited the acidosis-induced production of TBARS. The results suggest that the contribution of acidosis to peroxidative damages is probably of less importance in comparison to other cytotoxic mechanisms.     

Acidosis-induced modifications of high-affinity choline uptake by synaptosomes: Effects of pH readjustment

Acidosis (pH 6.0) led to significant decrease in high—affinity choline uptake by rat brain synaptosomes. The effects persisted following pH readjustment (7.4) of the incubation medium, consisting of decrease in both Km and Vmax of the affinity system. pH readjustment coincided with synaptosomal leakage of lactate dehydrogenase (LDH) and with instability of the synaptosomal suspension as evidenced from turbidity modifications of the preparation. LDH leakage occurred when acidosis was performed with lactic acid, whereas it was not seen following H₃PO₄ acidosis, probably because of the rapid diffusion of the protonated form of lactic acid across membranes. Turbidity modifications of the suspension were prevented by EDTA. The present results indicate that acidosis to pH level comparable to what is observed in brain ischemia is deleterious for cholinergic mechanisms. They also suggest that alkaline pH shifts that occur after blood reperfusion of ischemic brain tissue might be critical for the survival of cells.To whom to address reprint requests.     

Effects of a direct injection of liposoluble iron into rat striatum. Importance of the rate of iron delivery to cells

For a better understanding of the role of iron imbalance in neuropathology, a liposoluble iron complex (ferric hydroxyquinoline, FHQ) was injected into striatum of rats. The effects of two modalities of iron injections on brain damage, hydroxyl radical ( •OH) production (assessed by the salicylate method coupled to microdialysis) and tissue reactive iron level (evaluated ex vivo by the propensity of the injected structure for lipid peroxidation) were examined. Rapid injection of FHQ (10 nmoles of 5 mM FHQ pH 3 solution over 1-min period) but not that of corresponding vehicle led to extensive damage associated with increased tissue free iron level in the injected region. Conversely, neither lesion nor free iron accumulation was observed after slow FHQ injection (10 nmoles of a 100 μM FHQ pH 7 solution over 1-h period) as compared to corresponding vehicle injection. Production of •OH was induced by slow FHQ injection but not by rapid FHQ injection, probably as a result of in vivo abolition of iron-induced •OH formation by acid pH. Indeed, rapid injection of FAC pH 7 (ferric ammonium citrate, 5 mM in saline) was associated with •OH formation whereas rapid injection of FAC pH 3 did not. Our results identify the rate of iron delivery to cells as an important determinant of iron toxicity and do not support a major role for extracellular •OH in damage associated with intracerebral iron injection.     

Potential role of the neuropeptide CGRP in the induction of differentiation of rat hepatic portal vein wall

The media of the rat hepatic portal vein is composed of an internal circular muscular layer (CL) and an external longitudinal muscular layer (LL). These two perpendicular layers differentiate progressively from mesenchymal cells within the first month after birth. In this paper, we studied the development of calcitonin gene-related peptide (CGRP) innervation during post-natal differentiation of the vessel. We show that CGRP innervation is already present around the vessel at birth in the future adventitia but far from the lumen of the vessel. Progressively, CGRP immunoreactive fibers reached first LL then CL. CL by itself become only innervated at day 14 after birth. This corresponds to the time at which thick filaments (myosin) are visible in electron microscopy and desmin visualisable by immunocytochemistry. Furthermore, we provide evidence by autoradiography, that binding sites for CGRP are transiently expressed on the portal vein media at day 1 and 14 after birth. Vascular smooth muscle cells were transfected with constructs containing promoters for desmin or smooth muscle myosin heavy chain (smMHC). CGRP treatment of the cells significantly increased the expression of smMHC. Overall these results suggest that CGRP can potentially influence the differentiation of smooth muscle cells from the vessel wall.     

Human galectin 3 binding protein interacts with recombinant adeno-associated virus type 6

Recombinant adeno-associated viruses (rAAVs) hold enormous potential for human gene therapy. Despite the well-established safety and efficacy of rAAVs for in vivo gene transfer, there is still little information concerning the fate of vectors in blood following systemic delivery. We screened for serum proteins interacting with different AAV serotypes in humans, macaques, dogs, and mice. We report that serotypes rAAV-1, -5, and -6 but not serotypes rAAV-2, -7, -8, -9, and -10 interact in human sera with galectin 3 binding protein (hu-G3BP), a soluble scavenger receptor. Among the three serotypes, rAAV-6 has the most important capacities for binding to G3BP. rAAV-6 also bound G3BP in dog sera but not in macaque and mouse sera. In mice, rAAV-6 interacted with another protein of the innate immune system, C-reactive protein (CRP). Furthermore, interaction of hu-G3BP with rAAV-6 led to the formation of aggregates and hampered transduction when the two were codelivered into the mouse. Based on these data, we propose that species-specific interactions of AAVs with blood proteins may differentially impact vector distribution and efficacy in different animal models.     

The parasitic fly Nemorilla maculosa exploits host‐plant volatiles to locate the legume pod borer,Maruca vitrata

Nemorilla maculosa Meigen (Diptera: Tachinidae) is a solitary endoparasitoid of the legume pod borer, Maruca vitrata Fabricius (Lepidoptera: Crambidae), a key pest of cowpea, Vigna unguiculata (L.) Walp. (Fabaceae) in Africa. A colony of N. maculosa, introduced for experimental purposes from Taiwan to the laboratories of the International Institute of Tropical Agriculture (IITA) in Benin, was used for our studies. Olfactory reponses of N. maculosa to leaves of infested or uninfested cowpea and yellow peabush, Sesbania cannabina (Retz.) Pers. (Fabaceae), and to M. vitrata larvae were evaluated in a four‐arm olfactometer. For all combinations of odor sources, responses between naïve and oviposition‐experienced female flies did not differ. Nemorilla maculosa females were attracted by odors from uninfested leaves of yellow peabush and flowers of cowpea when compared with clean air, and they were attracted to plants damaged by M. vitrata with larvae removed. However, the female fly did not discriminate between odors from infested and uninfested plants. The parasitic fly N. maculosa proved well able to use volatile compounds from various host plants (peabush and cowpea) to locate its host, with a more pronounced attraction by the combination of host larvae and infested host plant parts. These findings are discussed in light of the prospective use of N. maculosa as a biological control agent against the legume pod borer.     

Effect of oxotremorine,physostigmine, and scopolamine on brain acetylcholine synthesis: A study using HPLC

The synthesis rate of brain acetylcholine (ACh) was estimated in mice following i.v. administration of [³H]choline (Ch). The measurements were performed 1 min after the tracer injection, using the [³H]ACh/[³H]Ch specific radioactivity ratio as an index of ACh synthesis rate. Endogenous and labeled Ch and ACh were quantified using HPLC methodology. Oxotremorine and physostigmine (0.5 mg/kg, i.p.) increased the steady state concentration of brain ACh by +130% and 84%, respectively and of Ch by +60% (oxotremorine); they decreased ACh synthesis by 62 and 55%, respectively. By contrast, scopolamine (0.7 mg/kg, i.p.) decreased the cerebral content of Ch by –26% and of ACh by –23% without enhancing the synthesis of ACh. The results show the utility of HPLC methodology in the investigation of ACh turnover.