The Hemileia vastatrix effector HvEC-016 suppresses bacterial blight symptoms in coffee genotypes with the SH 1 rust resistance gene.

A number of genes that confer resistance to coffee leaf rust (SH 1-SH 9) have been identified within the genus Coffea, but despite many years of research on this pathosystem, the complementary avirulence genes of Hemileia vastatrix have not been reported. After identification of H. vastatrix effector candidate genes (HvECs) expressed at different stages of its lifecycle, we established an assay to characterize HvEC proteins by delivering them into coffee cells via the type-three secretion system (T3SS) of Pseudomonas syringae pv. garcae (Psgc). Employing a calmodulin-dependent adenylate cyclase assay, we demonstrate that Psgc recognizes a heterologous P. syringae T3SS secretion signal which enables us to translocate HvECs into the cytoplasm of coffee cells. Using this Psgc-adapted effector detector vector (EDV) system, we found that HvEC-016 suppresses the growth of Psgc on coffee genotypes with the SH 1 resistance gene. Suppression of bacterial blight symptoms in SH 1 plants was associated with reduced bacterial multiplication. By contrast, HvEC-016 enhanced bacterial multiplication in SH 1-lacking plants. Our findings suggest that HvEC-016 may be recognized by the plant immune system in a SH 1-dependent manner. Thus, our experimental approach is an effective tool for the characterization of effector/avirulence proteins of this important pathogen.

c-FOS expression after hippocampal deep brain stimulation in normal rats.

OBJECTIVES: We studied the effects of Hip-deep brain stimulation (DBS) on the expression of the inducible transcription factor c-FOS in the brain of normal rats. MATERIALS AND METHODS: Ten Wistar rats were anesthetized, and nine were implanted with epidural and hippocampal electrodes for brain activity recording; one animal was used as sham. Bipolar stimulating electrodes were implanted in the left hippocampus. Three animals were used as control (implanted but not stimulated), one as sham (not implanted, not stimulated), and six as the study group. Stimulation was carried out using square wave pulses with 0.8V, 300 µsec, and 130 Hz (∼25µC/cm2) on the left hippocampus through the implanted bipolar hippocampal lead. Three animals were submitted to a one-hour and three to a six-hour stimulation session. Immunohistochemistry was employed to visualize c-FOS distribution in the rat's brain. The presence of seizures and electrocorticographic findings also were observed. RESULTS: In animals submitted to both one-hour or six-hour unilateral hippocampal stimulation sessions, there was a significant bilateral overexpression of c-FOS in the hippocampus proper, dentate gyrus, and hylus. In the CA1 and CA3 regions, although activation was bilateral, c-FOS hyperexpression prevailed at the stimulated side over time; this was not true for the hilar and dentate gyrus regions where a more symmetric activation occurred over time. A significant c-FOS activation occurred after one hour of Hip-DBS in the ipsilateral amygdala; there was no contralateral amygdala activation, and by six hours, no amygdala activation was noted. No c-FOS activation was noted in other brain areas. DISCUSSION: Our data showed that unilateral Hip-DBS was able to cause widespread and persistent bilateral activation of the normal rat limbic system, although in some, nuclei activation prevailed over the stimulated side. Cortical activation outside the limbic system was not noted. Our data represent a first approach to study the mechanistic paradigm involved in Hip-DBS.

Brain electrical activity after acute hippocampal stimulation in awake rats.

OBJECTIVE: We describe the electrocorticographic findings after hippocampal stimulation in normal awake rats. METHODS: Six male Wistar rats were implanted bilaterally with neocortical and hippocampal electrodes. The animals were submitted to hippocampal low- and high-frequency stimulation. RESULTS: Recruiting responses were seen in the ipsilateral hippocampus after unilateral low-frequency (6 Hz) hippocampal stimulation with low voltage (0.3 V). These recruiting responses could be seen at the contralateral hippocampus with slightly higher voltage (0.5 V) and over the ipsilateral neocortex with stimulation with 1.2 V. Bilateral neocortical recruiting responses were noted at stimuli voltage of 1.5 V. There were no recruiting responses after high-frequency stimulation (130 Hz). A dorsal column (DC) shift, characterized by baseline oscillation without brain activity modification, was noted in all animals after hippocampal stimulation with frequency higher than 60 Hz. The increase of stimulation frequency from 6 to 130 Hz (1.2 V, 300 µ sec) showed progressive reduction in the amplitude and disappearance of the time-locked recruiting responses, especially from around 60 Hz. CONCLUSIONS: Bilateral hippocampal and cortical recruiting responses were easily obtained in all animals after low-frequency hippocampal unilateral stimulation. High-frequency stimulation did not give rise to recruiting response, although a DC shift was noted. The fact that unilateral hippocampal stimulation might lead to bilateral limbic system modulation suggested that unilateral stimulation might be enough in many situations. Our findings suggested that high-frequency stimulation was more likely to be effective than low-frequency stimulation regarding the potential inactivation of the hippocampus. These findings might prove relevant to the determination of the adequate parameters for stimulation using hippocampal deep brain stimulation (DBS) in the future. An increase in our knowledge on the physiologic mechanisms underlying DBS might be translated into more rational clinical approaches.

Anticonvulsant, Anxiolytic and Antidepressant Properties of the ß-caryophyllene in Swiss Mice: Involvement of Benzodiazepine-GABAAergic, Serotonergic and Nitrergic Systems.

BACKGROUND: Central nervous system disorders such as anxiety, depression and epilepsy are characterized by sharing several molecular mechanisms in common and the involvement of the L-arginine/NO pathway in neurobehavioral studies with ß-caryophyllene is still little discussed. OBJECTIVES: One of the objectives of the present study was to demonstrate the anxiolytic behavioral effect of ß-caryophyllene (ß-CBP) in female Swiss mice, as well as to investigate the molecular mechanisms underlying the results obtained. METHODS: This study evaluated the neurobehavioral effects of ß-CBP using the open field test, rota- rod test, elevated plus maze test, novelty suppressed feeding test, tail suspension test and forced swim test, as well as pilocarpine, pentylenetetrazole and isoniazid-induced epileptic seizure models. RESULTS: The results demonstrated that the neuropharmacological activities of ß-CBP may involve benzodiazepine/GABAergic receptors, since the pre-treatment of ß-CBP (200 mg/kg) associated with flumazenil (5 mg/kg, benzodiazepine receptor antagonist) and bicuculline (1 mg/kg, selective GABAA receptor antagonist) reestablished the anxiety parameters in the elevated plus-maze test, as well as the results of reduced latency to consume food in the novelty suppressed feeding test. In addition to benzodiazepine/GABAergic receptors, the neuropharmacological properties of ß-CBP may be related to inhibition of nitric oxide synthesis, since pre-treatment with L-arginine (500-750 mg/kg) reversed significantly the anxiolytic, antidepressant and anticonvulsant activities of ß-CBP. CONCLUSION: The results obtained provide additional support in understanding the neuromolecular mechanisms underlying the anxiolytic, antidepressant and anticonvulsive properties of ß-CBP in female Swiss mice.