Cannabidiol in the prelimbic cortex modulates the comorbid condition between the chronic neuropathic pain and depression-like behaviour in rats: The role of medial prefrontal cortex 5-HT1A and CB1 receptors.

The prelimbic division (PrL) of the medial prefrontal cortex (mPFC) is a cerebral division that is putatively implicated in the chronic pain and depression. We investigated the activity of PrL cortex neurons in Wistar rats that underwent chronic constriction injury (CCI) of sciatic nerve and were further subjected to the forced swimming (FS) test and mechanical allodynia (by von Frey test). The effect of blockade of synapses with cobalt chloride (CoCl2), and the treatment of the PrL cortex with cannabidiol (CBD), the CB1 receptor antagonist AM251 and the 5-HT1A receptor antagonist WAY-100635 were also investigated. Our results showed that CoCl2 decreased the time spent immobile during the FS test but did not alter mechanical allodynia. CBD (at 15, 30 and 60 nmol) in the PrL cortex also decreased the frequency and duration of immobility; however, only the dose of 30 nmol of CBD attenuated mechanical allodynia in rats with chronic NP. AM251 and WAY-100635 in the PrL cortex attenuated the antidepressive and analgesic effect caused by CBD but did not alter the immobility and the mechanical allodynia when administered alone. These data show that the PrL cortex is part of the neural substrate underlying the comorbidity between NP and depression. Also, the previous blockade of CB1 cannabinoid receptors and 5-HT1A serotonergic receptors in the PrL cortex attenuated the antidepressive and analgesics effect of the CBD. They also suggest that CBD could be a potential medicine for the treatment of depressive and pain symptoms in patients with chronic NP/depression comorbidity.

Indomethacin attenuates mechanical allodynia during the organization but not the maintenance of the peripheral neuropathic pain induced by nervus ischiadicus chronic constriction injury.

The neurochemical mechanisms underlying neuropathic pain (NP) are related to peripheral and central sensitization caused by the release of inflammatory mediators in the peripheral damaged tissue and ectopic discharges from the injured nerve, leading to a hyperexcitable state of spinal dorsal horn neurons. The aim of this work was to clarify the role played by cyclooxygenase (COX) in the lesioned peripheral nerve in the development and maintenance of NP by evaluating at which moment the non-steroidal anti-inflammatory drug indomethacin, a non-selective COX inhibitor, attenuated mechanical allodynia after placing one loose ligature around the nervus ischiadicus, an adaptation of Bennett and Xie's model in rodents. NP was induced in male Wistar rats by subjecting them to chronic constriction injury (CCI) of the nervus ischiadicus, placing one loose ligature around the peripheral nerve, and a sham surgery (without CCI) was used as control. Indomethacin (2 mg/kg) or vehicle was intraperitoneally and acutely administered in each group of rats and at different time windows (1, 2, 4, 7, 14, 21, and 28 days) after the CCI or sham surgical procedures, followed by von Frey's test for 30 min. The data showed that indomethacin decreased the mechanical allodynia threshold of rats on the first, second, and fourth days after CCI (P<0.05). These findings suggested that inflammatory mechanisms are involved in the induction of NP and that COX-1 and COX-2 are involved in the induction but not in the maintenance of NP.

Indomethacin attenuates mechanical allodynia during the organization but not the maintenance of the peripheral neuropathic pain induced by nervus ischiadicus chronic constriction injury

The neurochemical mechanisms underlying neuropathic pain (NP) are related to peripheral and central sensitization caused by the release of inflammatory mediators in the peripheral damaged tissue and ectopic discharges from the injured nerve, leading to a hyperexcitable state of spinal dorsal horn neurons. The aim of this work was to clarify the role played by cyclooxygenase (COX) in the lesioned peripheral nerve in the development and maintenance of NP by evaluating at which moment the non-steroidal anti-inflammatory drug indomethacin, a non-selective COX inhibitor, attenuated mechanical allodynia after placing one loose ligature around the nervus ischiadicus, an adaptation of Bennett and Xie's model in rodents. NP was induced in male Wistar rats by subjecting them to chronic constriction injury (CCI) of the nervus ischiadicus, placing one loose ligature around the peripheral nerve, and a sham surgery (without CCI) was used as control. Indomethacin (2 mg/kg) or vehicle was intraperitoneally and acutely administered in each group of rats and at different time windows (1, 2, 4, 7, 14, 21, and 28 days) after the CCI or sham surgical procedures, followed by von Frey's test for 30 min. The data showed that indomethacin decreased the mechanical allodynia threshold of rats on the first, second, and fourth days after CCI (P<0.05). These findings suggested that inflammatory mechanisms are involved in the induction of NP and that COX-1 and COX-2 are involved in the induction but not in the maintenance of NP.

CB1 cannabinoid receptor-mediated anandamide signaling mechanisms of the inferior colliculus modulate the haloperidol-induced catalepsy.

The inferior colliculus (IC), a midbrain structure that processes acoustic information of aversive nature, is distinguished from other auditory nuclei in the brainstem by its connections with structures of the motor system. Previous evidence relating the IC to motor behavior shows that glutamatergic and GABAergic mechanisms in the IC exert influence on systemic haloperidol-induced catalepsy. There is substantial evidence supporting a role played by the endocannabinoid system as a modulator of the glutamatergic neurotransmission, as well as the dopaminergic activity in the basal nuclei and therefore it may be considered as a potential pharmacological target for the treatment of movement disorders. The present study evaluated if the endocannabinoid system in the IC plays a role in the elaboration of systemic haloperidol-induced catalepsy. Male Wistar rats received intracollicular microinjection of either the endogenous cannabinoid anandamide (AEA) at different concentrations (5, 50 or 100pmol/0.2µl), the CB1 cannabinoid receptor antagonist AM251 at 50, 100 or 200pmol/0.2µl or vehicle, followed by intraperitoneal (IP) administration of either haloperidol at 0.5 or 1mg/kg or physiological saline. Systemic injection of haloperidol at both doses (0.5 or 1mg/kg, IP) produced a cataleptic state, compared to vehicle/physiological saline-treated group, lasting 30 and 50min after systemic administration of the dopaminergic receptors non-selective antagonist. The midbrain microinjection of AEA at 50pmol/0.2µl increased the latency for stepping down from the horizontal bar after systemic administration of haloperidol. Moreover, the intracollicular administration of AEA at 50pmol/0.2µl was able to increase the duration of catalepsy as compared to AEA at 100pmol/0.2-µl-treated group. Intracollicular pretreatment with AM251 at the intermediate concentration (100pmol/0.2µl) was able to decrease the duration of catalepsy after systemic administration of haloperidol. However, neither the intracollicular microinjection of AM251 at the lowest (50pmol/0.2µl) nor at the highest (200pmol/0.2µl) concentration was able to block the systemic haloperidol-induced catalepsy. Furthermore, the intracollicular administration of AM251 at 100pmol/0.2µl was able to decrease the duration of catalepsy as compared to AM251 at 50pmol/0.2µl- and AM251 at 200pmol/0.2-µl-treated group. The latency for stepping down from the horizontal bar - induced by haloperidol administration - was decreased when microinjection of AEA at 50pmol/0.2µl was preceded with blockade of CB1 receptor with AM251 (100pmol/0.2µl). Our results strengthen the involvement of CB1-signaled endocannabinoid mechanisms of the IC in the neuromodulation of catalepsy induced by systemic administration of the dopaminergic receptors non-selective antagonist haloperidol.

Colletotrichum boninense Causing Anthracnose on Coffee Trees in Brazil.

In Brazil, dieback and necrosis of leaves and berries of coffee trees (Coffea arabica and C. canephora) are common symptoms of anthracnose disease caused by Colletotrichum gloeosporioides (Penz.) Sacc. In April 2010, these symptoms were observed in 100% of the plants from different coffee plantations in the Brazilian states of Espírito Santo and Bahia. Ten isolates were obtained from symptomatic leaves and berries from these areas. Of the 10 isolates, one had distinct conidial morphology with hyaline and ellipsoid conidia measuring 10 to 16 × 5.0 to 7.5 µm and melanized irregular or spatulated-shaped appressoria measuring 7.5 to 11.0 × 5.5 to 8.5 µm, formed either solitary or concatenated, which concurred with the conidia description of Colletotrichum boninense. In order to confirm the identity of this isolate, the internal transcribed spacer (ITS) rRNA region and the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene were sequenced (GenBank Accession Nos. JF683320 and JF331654, respectively) and compared to sequences from a database of C. boninense, confirming that the isolate was definitely C. boninense sensu lato, since it was exactly identical to other sequences in a large clade of isolates. To verify the pathogenicity of C. boninense in coffee and to compare the symptoms with those caused by C. gloeosporioides, leaves and berries were inoculated with the isolate of C. boninense and one representative isolate of C. gloeosporioides, both expressing the GFP (green fluorescent protein) gene. The isolates were grown for 7 days on potato dextrose agar and a conidial suspension (106 conidia × ml-1) was used to inoculate the organs, wounded and non-wounded, at different stages of development. In non-wounded organs, the conidial suspension was inoculated on the surface, and in leaves and berries used as control, the suspensions were substituted for sterile water. Leaves and berries were wounded with a sterilized needle and inoculated with 20 and 10 µl of the conidial suspension, respectively. Inoculated materials were incubated at 25°C and 100% relative humidity. The experiment was performed twice and evaluated daily for a week. No symptoms were observed on the control and non-wounded organs, while wounded organs exhibited typical anthracnose symptoms for both species. In berries, C. gloeosporioides consistently caused more severe symptoms at a faster rate than C. boninense. Both fungi caused necrosis in young but not old leaves. Typical acervuli were observed on the lesions and the fungus was successfully recovered from the inoculated tissues, which was confirmed by fluorescence microscopy, fulfilling Koch's Postulates. C. boninense has been identified as a pathogen causing anthracnose in a range of hosts worldwide. However, in Brazil, it has only been reported in pepper (Capsicum annuum) (3), passion fruit (Passiflora) (4), Hippeastrum (1) and in the medicinal plant Maytenus ilicifolia (2). To our knowledge, this is the first report of C. boninense associated with anthracnose of coffee trees in Brazil. Since the symptoms are similar to those caused by C. gloeosporioides, it can be stated that both species are associated with this disease in commercial coffee plantations in Brazil. Therefore, control strategies should consider the occurrence of C. boninense. References: (1) D. F. Farr et al. Mycol. Res. 110:1395, 2006. (2) S. A. Pileggi et al. Can. J. Microbiol. 55:1076, 2009. (3) H. J. Tozze et al. Plant Dis. 93:106, 2009. (4) H. J. Tozze et al. Australas. Plant Dis. Notes 5:70, 2010.

Lipoic acid effects on glutamate and taurine concentrations in rat hippocampus after pilocarpine-induced seizures / Efeitos do ácido lipóico nas concentrações de glutamato e taurina no hipocampo de ratos após convulsões induzidas por pilocarpina

Pilocarpine-induced seizures can be mediated by increases in oxidative stress and by cerebral amino acid changes. The present research suggests that antioxidant compounds may afford some level of neuroprotection against the neurotoxicity of seizures in cellular level. The objective of the present study was to evaluate the lipoic acid (LA) effects in glutamate and taurine contents in rat hippocampus after pilocarpine-induced seizures. Wistar rats were treated intraperitoneally (i.p.) with 0.9 percent saline (Control), pilocarpine (400 mg/kg, Pilocarpine), LA (10 mg/kg, LA), and the association of LA (10 mg/kg) plus pilocarpine (400 mg/kg), that was injected 30 min before of administration of LA (LA plus pilocarpine). Animals were observed during 24 h. The amino acid concentrations were measured using high-performance liquid chromatograph (HPLC). In pilocarpine group, it was observed a significant increase in glutamate content (37 percent) and a decrease in taurine level (18 percent) in rat hippocampus, when compared to control group. Antioxidant pretreatment significantly reduced the glutamate level (28 percent) and augmented taurine content (32 percent) in rat hippocampus, when compared to pilocarpine group. Our findings strongly support amino acid changes in hippocampus during seizures induced by pilocarpine, and suggest that glutamate-induced brain damage plays a crucial role in pathogenic consequences of seizures, and imply that strong protective effect could be achieved using lipoic acid through the release or decrease in metabolization rate of taurine amino acid during seizures.

As convulsões induzidas pela pilocarpina podem ser mediadas através do aumento do estresse oxidativo cerebral e das alterações na concentração dos aminoácidos. O presente estudo sugere que compostos antioxidantes podem produzir neuroproteção contra a neurotoxicidade em nível celular causada pelas convulsões. O objetivo deste estudo foi avaliar os efeitos do ácido lipóico (AL) no conteúdo de glutamato e taurina no hipocampo de ratos durante convulsões induzidas por pilocarpina. Ratos Wistar foram tratados por via intraperitoneal com solução salina 0,9 por cento (controle), pilocarpina (400 mg/kg, pilocarpina), AL (10 mg/kg) e com a associação de AL (10 mg/kg); 30 min após com pilocarpina (400 mg/kg), que foi injetada 30 min após a administração de AL (AL + pilocarpina). Os animais foram observados durante 24 horas. As concentrações de aminoácidos foram determinadas por HPLC. No hipocampo dos ratos do grupo pilocarpina foi observado um aumento significativo de 37 por cento na concentração de glutamato e uma diminuição de 18 por cento no nível de taurina, quando comparado ao grupo controle. O pré-tratamento com o antioxidante reduziu significativamente o nível de glutamato em 28 por cento e aumentou em 32 por cento os níveis de taurina no hipocampo dos ratos, quando comparado ao grupo pilocarpina. Nossos resultados sugerem que ocorrem alterações na concentração dos aminoácidos no hipocampo de ratos durante as convulsões induzidas por pilocarpina, e que o glutamato pode desempenhar um papel crucial na fisiopatologia das convulsões, e que o efeito protetor poderia ser alcançado com pré-tratamento com ácido lipóico, provavelmente pelo aumento da liberação ou redução da taxa de metabolização dos aminoácidos durante as convulsões.

Choline acetyltransferase and acetylcholinesterase activities are reduced in rat striatum and frontal cortex after pilocarpine-induced seizures.

In the present study we investigated the alterations on choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activities in rat striatum and frontal cortex caused by pilocarpine-induced seizures. Wistar rats were treated with 0.9% saline (i.p., control group), with the association of 0.9% saline (i.p.) plus pilocarpine (400mg/kg, i.p.), 30 min before of administration of saline (pilocarpine group). After the treatments all groups were observed for 1h. The ChAT and AChE activities were measured using spectrophotometric methods and the results compared to values obtained from saline-treated animals. In pilocarpine group was observed a significantly decreases in ChAT and AChE activities in striatum and frontal cortex of adult rats, when compared to control group. Results showed that during acute phase of seizures striatal and frontal cortex ChAT and AChE activities are diminished. Our findings suggest that seizures caused cognitive dysfunction and decreases of ChAT and AChE activities that might be related, at least in part, to the neurological problems presented by epileptic patients.

Study pharmacologic of the GABAergic and glutamatergic drugs on seizures and status epilepticus induced by pilocarpine in adult Wistar rats.

This work was designed to study the influence of drugs during seizures and status epilepticus (SE) induced by pilocarpine and mortality in adult rats. Glutamate (10 and 20 mg/kg), N-methyl-d-aspartate (NMDA, 5 and 10 mg/kg), ketamine (1.5 and 2.0 mg/kg), gabapentin (200 and 250 mg/kg), phenobarbital (50 and 100 mg/kg) and vigabatrin (250 and 500 mg/kg) were administered intraperitoneally, 30 min prior to pilocarpine (400 mg/kg, i.p.). The animals were observed (24 h) to determine: number of peripheral cholinergic signs, tremors, stereotyped movements, seizures, SE, latency to first seizure and number of deaths after pilocarpine treatment. NMDA and glutamate had pro-convulsive effects in both doses tested. Smaller and higher doses of these drugs no protected and increased pilocarpine-induced seizures and/or mortality. Gabapentin, vigabatrin, phenobarbital and ketamine protected against seizures and increased the latency to first seizure. Thus, these results suggest that caution should be taken in the selection of pharmacotherapy and dosages for patients with seizures and SE because of the possibility of facility the convulsive process toxicity, SE and the mortality of adult animals in this seizures model that is similar temporal lobo epilepsy in humans.

Effects of the vitamin E in catalase activities in hippocampus after status epilepticus induced by pilocarpine in Wistar rats.

Experimental manipulations suggest that in vivo administration of exogenous antioxidants agents decreases the concentration of free radical in the brain. Neurochemical studies have proposed a role for catalase in brain mechanisms responsible by development to status epilepticus (SE) induced by pilocarpine. The present study was aimed at was investigating the changes in catalase activities after pilocarpine-induced SE. Animals were treated with vitamin E (VIT E) 200 mg/kg (intraperitoneally (i.p.)) and, 30 min later, they received pilocarpine hydrochloride, 400 mg/kg, subcutaneous (s.c.) (P400). Other three groups received VIT E (200 mg/kg, i.p.), pilocarpine (400 mg/kg, s.c.) or 0.9% NaCl (control) alone. Animals were closely observed for behavioral changes, tremors, stereotyped movements, seizures, SE and death, for 24 h following the pilocarpine injection. The brains were dissected after decapitation. The results have shown that pilocarpine administration and resulting SE produced a significant increase in hippocampal catalase activity of (88%). In the group pre-treated which VIT E in hippocampal catalase activity was increase of 67% and 214% when compared with P400 and control group, respectively. Our results demonstrated a direct evidence of an increase in the activity of the hippocampal catalase of rat adults during seizure activity and after the pre-treated which VIT E that could be responsible by regulation of free radical levels during the establishment of SE.

Neuroanatomical approaches of the tectum-reticular pathways and immunohistochemical evidence for serotonin-positive perikarya on neuronal substrates of the superior colliculus and periaqueductal gray matter involved in the elaboration of the defensive behavior and fear-induced analgesia.

Deep layers of the superior colliculus, the dorsal periaqueductal gray matter and the inferior colliculus are midbrain structures involved in the generation of defensive behavior and fear-induced anti-nociception. Local injections of the GABA(A) antagonist bicuculline into these structures have been used to produce this defense reaction. Serotonin is thought to be the main neurotransmitter to modulate such defense reaction in mammals. This study is the first attempt to employ immunohistochemical techniques to locate serotonergic cells in the same midbrain sites from where defense reaction is evoked by chemical stimulation with bicuculline. The blockade of GABA(A) receptors in the neural substrates of the dorsal mesencephalon was followed by vigorous defensive reactions and increased nociceptive thresholds. Light microscopy immunocytochemistry with streptavidin method was used for the localization of the putative cells of defensive behavior with antibodies to serotonin in the rat's midbrain. Neurons positive to serotonin were found in the midbrain sites where defensive reactions were evoked by microinjection of bicuculline. Serotonin was localized to somata and projections of the neural networks of the mesencephalic tectum. Immunohistochemical studies showed that the sites in which neuronal perikarya positive to serotonin were identified in intermediate and deep layers of the superior colliculus, and in the dorsal and ventral columns of the periaqueductal gray matter are the same which were activated during the generation of defense behaviors, such as alertness, freezing, and escape reactions, induced by bicuculline. These findings support the contention that serotonin and GABAergic neurons may act in concert in the modulation of defense reaction in the midbrain tectum. Our neuroanatomical findings indicate a direct neural pathway connecting the dorsal midbrain and monoaminergic nuclei of the descending pain inhibitory system, with profuse synaptic terminals mainly in the pontine reticular formation, gigantocellularis nucleus, and nucleus raphe magnus. The midbrain tectum-gigantocellularis complex and midbrain tectum-nucleus raphe magnus neural pathways may provide an alternative output allowing the organization of the fear-induced anti-nociception by mesencephalic networks.

Effect of chronic intake of sweet substance on nociceptive thresholds and feeding behavior of Rattus norvegicus (Rodentia, Muridae).

The investigation of the influence of sweetened food on feeding behavior targeted to non-sucrose nutrients as well as the sensitivity to painful stimuli in isolated and grouped animals is the aim of the present work. The tail withdrawal latencies in the tail-flick test (a spinal reflex) were measured before and immediately after the treatment with tap water or sucrose (62, 125 or 250 g/l). Our findings suggest that: (a) The analgesic effect of sucrose intake depends on the concentration of sucrose solution and on the time during which the solution is consumed; (b) the most effective concentration of sucrose followed by antinociceptive effect was the one of 250 g/l in both isolated and grouped animals; (c) considering the individually caged rats, the intake of sucrose in the highest concentration (250 g/l) was the smallest as compared with the consumption of sucrose in more diluted solutions (62.5 and 125 g/l), but this higher sweetened solution was followed by antinociception; (d) animals treated with concentrated sucrose solution ate smaller quantities of pellets than animals treated with tap water; (e) tonic intake of highly concentrated sweet substance seems to be crucial for the increase of the nociceptive threshold in our model of sweet substance-induced antinociception.

Effect of the blockade of mu1-opioid and 5HT2A-serotonergic/alpha1-noradrenergic receptors on sweet-substance-induced analgesia.

RATIONALE: Sweet-substance-induced analgesia has been widely studied, and the investigation of the neurotransmitters involved in this antinociceptive process is an important way for understanding the involvement of the neural system controlling this kind of antinociception. OBJECTIVE: The aim of this study was to investigate the involvement of opioid and monoaminergic systems in sweet-substance-induced analgesia. METHODS: The present work was carried out in an animal model with the aim of investigating whether acute (24 h) or chronic (14 days) intake of a sweet substance, such as sucrose (250 g/l), is followed by antinociception. Tail withdrawal latencies in the tail-flick test were measured before and immediately after this treatment. Immediately after the recording of baseline values, independent groups of rats were submitted to sucrose or tap-water intake and, after chronic treatment, they were pretreated with intraperitoneal administration of (1) naltrexone at 0.5, 1, 2 or 3 mg/kg; (2) naloxonazine at 5, 10, 20 or 30 mg/kg; (3) methysergide at 0.5, 1, 2 or 3 mg/kg; (4) ketanserin at 0.5, 1, 2 or 3 mg/kg; or (5) physiological saline. RESULTS: Naltrexone and methysergide at two major doses decreased sweet-substance-induced analgesia after chronic intake of a sweet substance. These effects were corroborated by peripheral administration of naloxonazine and ketanserin. CONCLUSIONS: These data give further evidence for: (a) the involvement of endogenous opioids and a mu1-opioid receptor in the sweet-substance-induced antinociception; (b) the involvement of monoamines and 5HT2A serotonergic/alpha1-noradrenergic receptors in the central regulation of the sweet-substance-produced analgesia.

Opioid neurotransmission in the post-ictal analgesia: involvement of mu(1)-opioid receptor.

Pentylenetetrazol (PTZ), a non-competitive antagonist that blocks GABA-mediated Cl(-) flux, was used in the present work to induce seizures in animals. The aim of this work is to study the neurochemical basis of the antinociception induced by convulsions elicited by peripheral administration of PTZ (64 mg/kg). The analgesia was measured by the tail-flick test, in eight rats per group. Convulsions were followed by significative increase in the tail-flick latencies (TFL), for at least 120 min of the post-ictal period. Peripheral administration of naltrexone (5 mg/kg, 10 mg/kg and 20 mg/kg) caused a significant decrease in the TFL in seizing animals, as compared to controls. These data were corroborated with peripheral administration of naloxonazine (10 mg/kg and 20 mg/kg), a mu(1)-opioid blocker, in the same doses used for non-specific antagonist. These results indicate that endogenous opioids may be involved in the post-ictal analgesia. The involvement of mu(1)-opioid receptor was also considered.

Psychopharmacological evidences for the involvement of muscarinic and nicotinic cholinergic receptors on sweet substance-induced analgesia in Rattus norvegicus.

In order to investigate the effects of sweet substance intake on pain modulation, male albino Wistar rats weighing 180-200 g received either tap water or sucrose solutions (250 g/l) for 14 days as their only source of liquid. Each rat consumed an average of 15.6 g sucrose/day. Their tail withdrawal latencies in the tail-flick test (probably a spinal reflex) were measured immediately before and after this treatment. An analgesia index was calculated from the withdrawal latencies before and after treatment. The index (mean +/- SEM, N = 8) for the groups receiving sucrose solution plus saline (NaCl; 0.9%) for 14 days was 0.70 +/- 0.01. Atropine (1 and 2 mg/kg)-treated rats (N = 8) after intake of sucrose exhibited an analgesia index of 0.39 +/- 0.09 and 0.39 +/- 0.08, respectively, while mecamylamine (1 and 2 mg/kg)-treated rats (N = 10) after intake of sucrose had an index of -0.02 +/- 0.07 and 0.03 +/- 0.07, respectively. These results indicate that the effect of sucrose intake on nociceptive thresholds is controlled by neurotransmission of acetylcholine and depends on the nicotinic cholinergic receptors for its major analgesic effect, although muscarinic receptors were also involved in this antinociceptive process.

Use of a single method in the extraction of the seed storage globulins from several legume species. Application to analyse structural comparisons within the major classes of globulins.

In this study, a single, improved methodology was used to extract, fractionate and purify the 11S (legumin-type or related to the alpha-conglutin from Lupinus albus L.), 7S (vicilin-type or related to the beta-conglutin from L. albus) and 2S (related to the gamma-conglutin from L. albus) families of proteins from eight legume species: L. albus, Glycine max (L.) Merr., Pisum sativum L., Vicia faba L., Cicer arietinum L., Phaseolus vulgaris L., Lens culinaris Med. and Arachis hypogaea L. The sedimentation coefficients obtained varied from 1.9 to 8.1 for the gamma-conglutin-related proteins, from 5.1 to 10.5 for the beta-conglutin-related proteins and from 12.0 to 14.9 for the alpha-conglutin-related globulins. The gamma-conglutin-related proteins is the most heterogeneous group. Antibodies produced against each type of gamma-conglutin polypeptide chain recognize the other polypeptide chain as well as other polypeptides in the corresponding globulins from all species examined. The 7S globulins are typically composed of a large number of polypeptides, covering a wide range of molecular masses (10 to 70 kD). The presence of disulphide bonds is apparently absent and the occurrence of glycopolypeptides is not widespread. Finally, the 11S globulins are characteristically formed by a limited number of polypeptides that may be divided into a lighter group (20-25 kD) and a heavier group (35-50 kD). The presence of disulphide bonds is apparently widespread but the occurrence of glycopolypeptides seems to be relatively rare. Both the 7S family and the 11S globulins studied by immunoblotting exhibit a low level of structural similarity.