ω-Conotoxina MVIIC e células-tronco mesenquimais promovem recuperação motora em ratos Wistar após trauma medular agudo / ω-Conotoxin MVIIC and mesenchymal stem cells promote motor recovery in Wistar rats after acute spinal cord injury]

O objetivo deste estudo foi avaliar o efeito da ω-conotoxina MVIIC e das células-tronco mesenquimais (CTM) de forma isolada e sua associação nos ratos submetidos ao trauma medular agudo (TMA). Trinta Rattus novergicus, linhagem Wistar, três meses de idade, foram distribuídos igualmente em cinco grupos experimentais: controle negativo (CN), controle positivo (CP), ω-conotoxina MVIIC (MVIIC), células-tronco mesenquimais da medula óssea (CTM-MO) e associação (MVIIC + CTM-MO). O grupo CN foi submetido à laminectomia sem trauma medular, e os grupos CP, MVIIC, CTM-MO e MVIIC + CTM-MO foram submetidos ao trauma medular contusivo. O grupo CP recebeu, uma hora após o TMA, 10µL de PBS estéril, e os grupos MVIIC e MVIIC + CTM-MO receberam 10µL de PBS contendo 20pmol da ω-conotoxina MVIIC, todos por via intratecal. Os grupos CTM-MO e MVIIC + CTM-MO receberam, 24 horas após, 1x106 de CTM via intravenosa. Avaliou-se a recuperação da função locomotora até o sétimo dia pós-trauma. Os animais tratados com MVIIC + CTM-MO obtiveram recuperação motora após o trauma medular agudo (P<0,05). Conclui-se que essa associação apresentou efeito neuroprotetor com melhora na função locomotora em ratos Wistar.(AU)

The objective of this study was to evaluate the effect of isolated ω-conotoxin MVIIC and mesenchymal stem cells (MSCs) and its association in rats submitted to acute spinal cord injury (SCI). Thirty Rattus norvegicus, Wistar strain, three-month-old rats were randomly distributed in five experimental groups with six animals: negative control (CN), positive control (CP), ω-conotoxin MVIIC (MVIIC), bone marrow mesenchymal stem cells (CTM-MO) and the association (MVIIC + CTM-MO). The CN group underwent laminectomy without spinal cord trauma, and groups CP, MVIIC, CTM-MO and MVIIC + CTM-MO were submitted to contusive spinal cord trauma. The CP group received 10µl of PBS one hour after SCI, and groups MVIIC and MVIIC + CTM-MO received 10µl of PBS containing 20pmol of ω-conotoxin MVIIC, both intrathecally. Groups CTM-MO and MVIIC + CTM-MO received 1x106 of MSCs intravenously 24 hours later. The recovery of locomotor function was evaluated up to seven days post-injury. The animals treated with MVIIC + CTM-MO obtained motor recovery after SCI (P<0.05). It is concluded that this association showed neuroprotective effect with improvements in locomotor function in Wistar rats.(AU)

Increased levels of glutamate in the central nervous system are associated with behavioral symptoms in experimental malaria

Cerebral malaria (CM) is a severe complication resulting from Plasmodium falciparum infection. This condition has been associated with cognitive, behavioral and motor dysfunctions, seizures and coma. The underlying mechanisms of CM are incompletely understood. Glutamate and other metabolites such as lactate have been implicated in its pathogenesis. In the present study, we investigated the involvement of glutamate in the behavioral symptoms of CM. Seventeen female C57BL/6 mice (20-25 g) aged 6-8 weeks were infected with P. berghei ANKA by the intraperitoneal route using a standardized inoculation of 10(6) parasitized red blood cells suspended in 0.2 mL PBS. Control animals (N = 17) received the same volume of PBS. Behavioral and neurological symptoms were analyzed by the SmithKline/Harwell/Imperial College/Royal Hospital/Phenotype Assessment (SHIRPA) battery. Glutamate release was measured in the cerebral cortex and cerebrospinal fluid of infected and control mice by fluorimetric assay. All functional categories of the SHIRPA battery were significantly altered in the infected mice at 6 days post-infection (dpi) (P ≤ 0.05). In parallel to CM symptoms, we found a significant increase in glutamate levels in the cerebral cortex (mean ± SEM; control: 11.62 ± 0.90 nmol/mg protein; infected at 3 dpi: 10.36 ± 1.17 nmol/mg protein; infected at 6 dpi: 26.65 ± 0.73 nmol/mg protein; with EGTA, control: 5.60 ± 1.92 nmol/mg protein; infected at 3 dpi: 6.24 ± 1.87 nmol/mg protein; infected at 6 dpi: 14.14 ± 0.84 nmol/mg protein) and in the cerebrospinal fluid (control: 128 ± 51.23 pmol/mg protein; infected: 301.4 ± 22.52 pmol/mg protein) of infected mice (P ≤ 0.05). These findings suggest a role of glutamate in the central nervous system dysfunction found in CM.

Heterologous expression of an insecticidal gene from the armed spider (Phoneutria nigriventer)

Insect-pests are global problems that cause severe damage to crop plants, and their control is commonly based on chemical insecticides. However, negative effects of pesticides on the environment and human health emphasize the necessity to develop alternative methods for insect-pest control. In the present study, a gene coding for the insecticidal peptide TX4(6-1) of the Brazilian armed spider (Phoneutria nigriventer) was cloned in fusion with maltose binding protein (MBP) and expressed in Escherichia coli. The affinity purified protein MBP-GlyTX4 was cleaved with the Xa factor and used for a bioassay against Spodoptera frugiperda and rabbit immunization. Five micrograms GlyTX4 protein injected into the hemocoel of larvae and abdominal cavity of adults produced trembling and uncoordinated movements immediately after injection and all adult insects died after 12h. After two days, larvae became paralyzed and the epidermal color changed to dark brown. Furthermore, the development stage was prolonged for two weeks. Alternatively, slices of maize leaves were imbibed with 15 micrograms of the recombinant protein cleaved with the Xa factor and used as diet for larvae. In this experiment, all larvae died in about 30 minutes. Polyclonal antibodies anti-MBP-GlyTX4 were effective for recognizing MBP and GlyTX4 in whole cell extract from E. coli expressing the recombinant protein.

Use of fluorescent probes to follow membrane traffic in nerve terminals

Optical tracers in conjunction with fluorescence microscopy have become widely used to follow the movement of synaptic vesicles in nerve terminals. The present review discusses the use of these optical methods to understand the regulation of exocytosis and endocytosis of synaptic vesicles. The maintenance of neurotransmission depends on the constant recycling of synaptic vesicles and important insights have been gained by visualization of vesicles with the vital dye FM1-43. A number of questions related to the control of recycling of synaptic vesicles by prolonged stimulation and the role of calcium to control membrane internalization are now being addressed. It is expected that optical monitoring of presynaptic activity coupled to appropriate genetic models will contribute to the understanding of membrane traffic in synaptic terminals.

Effect of gamma interferon and interleukin 10 on phosphoinositol turnover by human neutrophils in vitro

We evaluated the levels of inositolmono-(IP1), di-(IP2), tri- (IP3) and tetraphosphates (IP4) in human neutrophils (N) stimulated with gamma interferon (IFN-gamma) (200 mul from a pool of cell culture supernatant obtained from 1 x 10(7) PHA-primed peripheral blood mononuclear cells (30-60 min at 37 degrees Celsius, 5 per cent CO2) in the presence or in the absence of interleukin 10 (IL-10) (10 mug/10mul). The results, reported as mean + SEM cpm, showed that IFN-gamma induced a significant increase only in the IP3 level (N + medium = 1.413 + 172 and N + IFN-gamma = 8,875 + 832). However, this activation mediated by IFN-gamma was blocked partially in the presence of IL-10 (N + IFN-gamma + IL-10 = 2,430 + 239) (P<0.05). Interleukin 10 alone did not induce significant alterations in the content of IP1 (1,203 + 123), IP2 (1,880 + 163), IP3 (938 + 102) or IP4 (2,403 + 345) when compared to the respective controls in the absence of IL-10 (IP1 = 1,625 + 132; IP2 = 1,343 + 149; IP3 = 1,413 + 172 and IP4 = 3,281 + 234). We also demonstrated the inhibitory effect of IL-10 on chemoluminescence generation by human neutrophils during phagocytosis of opsonized particles (OZ). Chemoluminescence generation was enhanced by IFN-gamma (N + OZ = 42.8 + 3.9 and N + OZ + IFN-gamma = 66.5 + 4.3) and this effect was reduced by IL-10 (N + OZ + IFN-gamma + IL-10 = 37.6 + 5.1). These data suggest that IL-10 modulates the neutrophil response and may be important for the development of new treatments of inflammatory injury.

The effect of crotoxin on the release of acetylcholine and lactate dehydrogenase from rat brain cortical slices

We have studied the effects of crotoxin, the neurotoxin of the South American rattlesnake Crotalus durissus terrificus, ,on the release of acetylcholine and lactate dehydrogenase from rat brain cortical slices. Crotoxin enhances the release of [3H]-acetylcholine from cortical slices (control values 92.8 ñ 5.9 and 150.3 ñ 11.7 DPM/mg and crotoxin valuesw 199.1 ñ 7.0 and 336.0 ñ 26.0 DPM/mg at 60 and 120 min incubation, respectively) in parallel with the release of lactate dehydrogenase (control values 50.4 ñ 16.8 and 80.3 ñ 19.5 U/mg and crotoxin values 162.5 ñ 39.1 and 355.7 ñ 38.2 U/mg, at 120 min incubation, respectively). Both effects are markedly reduced when substituting Sr2+ for Ca2+ in the incubation medium. It is concluded that the phospholipase activity of crotoxin is responsible for the observed effects

Correlation between the lack of mononuclear cell cytotoxicity against schistosomula in vitro with schistosomiasis

A comparison was made of the reactivity of mononuclear cells from subjects and from S. mansoni-infected patients. The following parameters were evaluated: 1) ability of mononuclear cells to kill schistosoma in the presence of complement; 2) [3H]-inositol incorporation into phosphatidylinositol (PI) and the rate of inositolphosphates (IPx) released. Cells from normal subjects, but not from S. mansoni infected patients, were able to kill schistosomula in vitro. A decrease in inositolpolyphosphates (IPx) was observed for phytohemagglutinin (PHA)-stimulated mononuclear cells from infected patients when compared with mononuclear cells from normal subjects after 24 h of incubation. The results suggest that the reactivity of mononuclear cells from infected patients is altered under conditions of nonspecific stimulation with PHA when compared with normal cells

Effect of tityustoxin and quabain on protein phosphorylation in a crude synaptosome fraction

1. The effect of titiyustoxin (TsTX) and ouabain on the incorporation of 32P into a protein of the same apparent molecular weight as synapsin I it described. 2. Tityustoxin-stimulated protein phosphorylation in a crude synaptosome fraction increased up to a concentration of 3.0 micron time of 15 s. 3. Trifluoperazizne (100 micronM) inhibited, while trifluoperazine sulphoxide (100 micronM) did not alter the effect on the protein phosphorylation induced by tityustoxin. 4. Unlike tityutixin, ouabain (100 micronM) hada no effect on protein phosphorylation even after incubation up to 20 min. 5. Ouabain at at 10 micronM, a concentration having no effect on rates of respiration and ATP hydrolysis in brain cortical slices, also had no effect on protein phosphorylation

Gastric acetylcholine and histamine content of normal and Trypanosoma cruzi - infected rats

The consequences of acute Trypanosoma cruzi infection for acetylcholine and histamine levels in gastric wall and for mast cells of the stomach were studied in rats. Intraperitoneal infection with 4,000 trypomastigotes/g of a Y strain of Trypanosoma cruzi led to a 4-fold decrease in gastric acetylcholine level and to a 57 - and 15-fold increase in histamine content in the membranous and glandular regions of the rat stomach, respectively. Infection of rats with Trypanosoma cruzi also induced a 2- and 4-fold increase in mast cell numbers in the membranous and glandular regions of the muscle layer of the gastric wall, respectively, and a ganglionic inflammatory reaction with predominance of mononuclear cells. We conclude that in acutely Trypanosoma cruzi-infected rats, the reduction of acetylcholine content is due to gastric denervation and that the histamine increase might be secondary to gastric denervation and/or to an increase in the number of mast cells of the gastric wall

Changes in choline acetyltransferase activity of rat tissues during Chagas' disease

1. The activity of choline acetyltransferase (CAT) measured by a radilabeling method was significantly reduced in the heart, submandibular gland and esophagus of rats 20 days after inoculation with Trypanosoma cruzi (Y strain). 2. Normal enzyme activity was recovered in all these organs 100 days after inoculation. 3. In the transcerse colon, no change, 30% reduction and normal activity were found at days 20, 100 and 430 of infection, respectively. 4. The data indicate recovery of parasympathetic function in experimental Chagas' disease. Axonal regrowth and sprouting are discussed as possible candidates for the recovery mechanisms