Genotoxicity of the medicinal plant Maytenus robusta in mammalian cells in vivo.

Plants belonging to the Celastraceae family have been used in traditional medicine for their analgesic, anti-inflammatory and anti-ulcerogenic properties, among others. Maytenus ilicifolia is the principal species of this family, and is used in the treatment of gastric ulcers. However, owing to its inadequate management in Brazil, the species is becoming extinct and is being substituted with Maytenus robusta, which also displays gastroprotective activity. The aim of this study was to evaluate the genotoxic effects of M. robusta hydroalcoholic extract in vivo, using the comet assay and micronucleus test. Three doses (50, 250 and 500 mg/kg body weight) were administered to mice orally 2 times at 24-h intervals. Cytotoxicity was assessed by scoring 200 consecutive total polychromatic and normochromatic erythrocytes to calculate their ratio. Parametric (analysis of variance/Tukey) and non-parametric (Kruskal-Wallis/Dunn post hoc) tests were used to evaluate the results according to the nature of the data distribution. The results showed a significant increase in the frequency of DNA damage on leukocytes at the 2 higher doses tested, but the extract did not enhance micronucleus frequency in bone marrow cells. Our findings showed that after 48 h of treatment, M. robusta hydroalcoholic extract had weak genotoxic effects but no clastogenic effects in mice cells.

Toxicity assessment of metabolites of fungal biocontrol agents using two different (Artemia salina and Daphnia magna) invertebrate bioassays.

Fungal biocontrol agents (BCAs) have been marketed for control of crop pests, weeds, and diseases. However, BCAs may produce toxic metabolites, whose presence in the formulated products, in the crops and in the environment should be considered along with the associated risk. Two invertebrate models, viz. Artemia salina and Daphnia magna were used to assess the acute toxicity of seven BCA metabolites, characterized by different chemical nature and mode of action, namely alamethicin (ALA), paracelsin (PCS), antiamoebin (AAM), gliotoxin (GTX), destruxin A (DA), oosporein (OOS), and elsinochrome A (EA). The two invertebrates were very sensitive to all the metabolites examined, except OOS. The LC50s after 24 and 36 h exposures showed the following toxicity ranks: A. salina, DA > ALA > EA > GTX > AAM > PCS (LC50s ranging from 9.78 to 40.84 microg/ml at 24 h and from 2.92 to 18.56 microg/ml at 36 h); D. magna, DA > GTX = EA > ALA > PCS > AAM (LC50s ranging from 0.20 to 24.41 microg/ml at 24h and from 0.16 to 11.98 microg/ml at 36 h). LC50 of OOS to D. magna increased dramatically in 36 h exposure, compared to 24 h exposures (5.84 and 68.40 microg/ml, respectively). A. salina and D. magna proved to be suitable models for rapid and inexpensive screening of toxicity of BCAs at an early stage of product development.

Linked activation of nitric oxide synthase and cyclooxygenase in peripheral monocytes of asymptomatic migraine without aura patients.

Many reports indicate that nitric oxide (NO) could be involved in migraine without aura (MWA), an extremely diffuse clinical event. Since monocyte may be a relevant source of NO, we analysed monocyte activation in MWA patients, in a period in which they were free of symptoms. NO basal production by MWA peripheral monocytes was significantly higher than in healthy subjects (91.25+/-8.6 microM/10(6) cells vs. 22.6+/-3.2 microM/106 cells). Interestingly, even the release of prostaglandin E2 (PGE2), was higher in MWA patients than in healthy subjects (3137+/-320 pg/10(6) cells vs. 1531+/-220 pg/10(6) cells). The incubation of monocytes from healthy subjects and MWA patients with N-nitro-L-arginine methyl ester caused a marked decrease of both NO and PGE2 release. We hypothesise that NOS and cyclooxygenase pathways in monocytes are linked and are, in MWA patients, up-regulated, even in a symptoms-free period. NO and PGE2 hyperproduction could therefore be involved in the neurovascular modifications leading to migraine attacks.

Concomitant activation of Gi protein-coupled receptor and protein kinase C or phospholipase C is required for platelet aggregation.

It has recently been suggested that the concomitant activation of two distinct G protein-coupled receptors (G(i) and G(q)) is essential for platelet aggregation: in fact, the thromboxane A2 synthetic agonist, U46619, which causes the selective activation of Gq, is not able to elicit fibrinogen receptor exposure unless ADP or epinephrine is present. In the present study we demonstrate that a direct Gq activation is not required for platelet aggregation and that the activation of an enzyme downstream of Gq, such as phospholipase C (PLC) or protein-kinase C (PKC), is sufficient for such a process. In fact, platelet aggregation occurred in response to the snake venom toxin convulxin, which activates the PLC isoform PLCgamma2 or to cytosolic PKC activator phorbol 12-myristate 13-acetate (PMA) provided a Gi protein-coupled receptor was activated by ADP or epinephrine. The evidence that the PKC inhibitor, Ro 31-8220 did not suppress platelet aggregation in response to convulxin plus ADP or epinephrine led us to conclude that PLC and PKC are both involved in platelet aggregation, although not concomitantly, provided a Gi protein-coupled receptor is activated.

Discrete and continuous planning of hand movements and isometric force trajectories.

We have previously demonstrated that, in preparing themselves to aim voluntary impulses of isometric elbow force to unpredictable targets, subjects selected default values for amplitude and direction according the range of targets that they expected. Once a specific target appeared, subjects specified amplitude and direction through parallel processes. Amplitude was specified continuously from an average or central default; direction was specified stochastically from one of the target directions. Using the same timed response paradigm, we now report three experiments to examine how the time available for processing target information influences trajectory characteristics in two-degree-of-freedom forces and multijoint movements. We first sought to determine whether the specification of force direction could also take the form of a discrete stochastic process in pulses of wrist muscle force, where direction can vary continuously. With four equiprobable targets (two force amplitudes in each of two directions separated by 22 degrees or 90 degrees), amplitude was specified from a central default value for both narrow and wide target separations as a continuous variable. Direction, however, remained specified as a discrete variable for wide target separations. For narrow target separations, the directional distribution of default responses suggested the presence of both discrete and central values. We next examined point-to-point movements in a multijoint planar hand movement task with targets at two distances and two directions but at five directional separations (from 30 degrees to 150 degrees separation). We found that extent was again specified continuously from a central default. Direction was specified discretely from alternative default directions when target separation was wide and continuously from a central default when separation was narrow. The specification of both extent and direction evolved over a 200-ms time period beginning about 100 ms after target presentation. As in elbow force pulses, extent was specified progressively in both correct and wrong direction responses through a progressive improvement in the scaling of acceleration and velocity peaks to the target. On the other hand, movement time and hand path straightness did not change significantly in the course of specification. Thus, the specification of movement time and linearity, global features of the trajectories, are given priority over the specific values of extent and direction. In a third experiment, we varied the distances between unidirectional target pairs and found that movement extent is specified discretely, like direction, when the disparity in distances is large. The implications of these findings for contextual effects on trajectory planning are discussed. The independence of extent and direction specification and the prior setting of response duration and straightness provide critical support for the hypothesis that point-to-point movements are planned vectorially.

Reaching movements: concurrency of continuous and discrete programming.

The present study was undertaken to investigate further how human subjects prepare default parameters of reaching movements. Two qualitatively different modes for setting default values of direction are possible (continuous and discrete mode) and there is a threshold visual target separation for continuous or discrete programming. The most prominent finding is that the continuous and discrete modes of programming can be used concurrently.

Reaching movements: programming time course is independent of choice number.

The present study was undertaken to assess the effect of varying the number of choices on the time needed by human subjects to program the correct direction of reaching arm movements. Subjects were required to reach for visual targets presented in two, four or eight different directions. In none of these subjects was the time needed to program response direction correctly prolonged by increasing the number of choices. In the present experimental situation, the time course of the process by which information derived from a visual target is used to accurately set the direction of reaching arm movements is uninfluenced by the number of possible targets.

Parallel direction and extent specification of planar reaching arm movements in humans.

The present study was undertaken to clarify further whether the programming of direction and extent of the same motor trajectory is accomplished by parallel or serial processing channels. We reasoned that if direction and distance are independently specified, then the time course of each process should not be influenced by the need for simultaneously specifying the other parameter. If direction and distance are specified in a serial order, the need to specify two parameters rather than only one should prolong the process of response specification. For this purpose experiments were run on the same six subjects, using our time-response paradigm and the data obtained were compared. To avoid any possible effect attributable to the Hick and Hyman law, the number of targets was maintained invariant and equal to four. Our major finding is that the time course of directional specification of reaching movements is not influenced by the need for simultaneously specifying extent. This is consistent with our view of parameterization of planning and executing movements, in which the two parameters (direction, extent) can be specified in parallel.

Photodynamic therapy: a 5-year study of its effectiveness in the treatment of posterior uveal melanoma, and evaluation of haematoporphyrin uptake and photocytotoxicity of melanoma cells in tissue culture.

A human melanoma cell line RVH-421 which selectively demonstrates melanin synthesis when cultured in L15 Leibowitz medium but not in RPMI medium was used as a model to examine haematoporphyrin derivative (HPD) uptake and the photocytotoxicity of photodynamic therapy (PDT). Confocal scanning microscopy and extraction fluorometry showed HPD uptake in both non-pigmented and pigmented melanoma cells. Phototoxicity was determined by incubating pigmented and non-pigmented monolayer cells with HPD, exposing them to variable periods of white fluorescent light and calculating the number of viable cells in the samples relative to the controls. Both the non-pigmented and pigmented melanoma cells were affected by light-dependent cytotoxicity which was greater in the non-pigmented cells. Melanin or other substances may reduce the photo-oxidative effects of PDT. Posterior uveal melanomas in 36 patients were treated with PDT with the longest duration of tumour control being 6.5 years. Kaplan-Meier survival analysis showed that 76% of melanomas were not growing at the end of the first year, declining to 62% at the end of the second year, with 38% showing no signs of growth at the end of the fifth year. No eyes were lost as a result of PDT. Cox's hazards analysis showed that the degree of tumour pigmentation and patient age at therapy significantly influence the tumour response to PDT.

Trajectory control in targeted force impulses. VII. Independent setting of amplitude and direction in response preparation.

We have previously shown that when aiming impulses of force to unpredictable flexion and extension targets, subjects prepare themselves to respond by preselecting a default amplitude and direction. In our preceding study, where flexion and extension target ranges were symmetrical, subjects prepared a single default amplitude near the center of each of the two ranges and selected a default direction arbitrarily (Favilla, Hening et al. 1989). Following target presentation, amplitude and direction were specified gradually and in parallel. By using an experimental paradigm in which target amplitudes had unequal probabilities in only one of the two directions, we no sought to determine to what extent the default amplitude prepared by subjects is dependent on the default direction that is selected for a given trial. Five normal subjects produced isometric elbow flexion and extension force impulses to match four targets requiring responses of two amplitudes in each direction. Flexion and extension targets were of the same size. In all biased probability conditions, targets were presented in unpredictable order and, in one direction, the probability of occurrence of the targets was biased (80% versus 20%). In the other direction, the two targets were equiprobable. To maximize the effect of the default settings on response parameters, we required subjects to initiate their response in synchrony with a predictable tone occuring less than a reaction time subsequent to the presentation of the visual target. Such a short interval was chosen to disclose the default parameters selected by limiting the influence of the target just presented on the amplitude and direction of the subjects' responses.(ABSTRACT TRUNCATED AT 250 WORDS)

Trajectory control in targeted force impulses. VI. Independent specification of response amplitude and direction.

The preceding study of this series (Hening, Favilla and Ghez 1988) examined the time course of the processes by which human subjects use information from a target to set the amplitude of an impulse of isometric elbow force. In that study, subjects were provided with separate cues to time response initiation and to inform them of the required amplitude of the response. When the time between target presentation and response initiation was too brief for them to incorporate information from the target, subjects produced default responses whose amplitudes reflected their prior experience. At longer latencies, subjects specified response amplitude with a gradual time course, starting earlier and ending later than an average reaction time. The present study now examines how two distinct response features, amplitude and direction, are specified following presentation of a target. We sought to answer three main questions. What are the features of responses that are produced before target information is available? Are direction and amplitude specified serially or in parallel? Does the specification of one response feature interfere with the specification of the other? Six normal subjects were studied. They were trained to initiate impulses of isometric elbow force in synchrony with the last of a predictable series of regular tones. The amplitudes and directions were to match those of visual targets requiring flexions or extensions with one of three amplitudes. The targets were presented at random times (0-400 ms) before the last tone. Target directions and amplitudes were either predictable (simple condition) or unpredictable (choice condition). In the simple condition, response amplitudes and directions were independent of the interval between target presentation and response onset (S-R interval). In the choice condition, both amplitude and direction varied with the S-R interval. At short S-R intervals (less than 100 ms), the direction of the subjects' responses was not related to that of the target. The amplitudes of the responses were near the centers of the two target ranges. With increasing S-R intervals, the proportion of correct direction responses gradually increased. Over the same range of S-R intervals, the amplitudes of both right and wrong direction responses to the different targets separated and converged on their respective target amplitudes. Specification of both direction and amplitude was complete at S-R intervals greater than 300 ms. The time course of amplitude specification in this bidirectional paradigm was prolonged over that in a paradigm where response direction was predictable.(ABSTRACT TRUNCATED AT 400 WORDS)

Gradual specification of response amplitude in human tracking performance.

These experiments examine how human subjects use information from a target to trigger a response and to specify its trajectory. We first determined if response initiation is predicated on the prior specification of response amplitude by examining the latencies and trajectories of impulses of isometric elbow flexion aimed to one of three visual targets. We varied target predictability (simple versus choice), the urgency with which the response was required, and the level of practice. With practice, subjects could respond to unpredictable targets with the same latency as to predictable ones; the range of response amplitudes was, however, always constricted. This central tendency bias disappeared when subjects were allowed long latencies to respond to the target, suggesting that with urgency, subjects can respond before specification is complete. To determine the time course of specification, the subjects were trained to initiate force impulses in synchrony with the last of a series of predictable tones. They also attempted to match the amplitude of their force impulses to one of three unpredictable visual targets presented at randomly varying times (50-400 ms) prior to the synchronizing tone. At the shortest stimulus-response intervals, before target information could be processed, the amplitudes of responses to all targets were clustered around that of the middle-sized target. Then, as the stimulus-response interval increased, response amplitudes gradually converged upon their specific targets. Specification started at stimulus-response intervals of about 100 ms and extended until about 350 ms.(ABSTRACT TRUNCATED AT 250 WORDS)

Trajectory control in targeted force impulses. V. Gradual specification of response amplitude.

This study was undertaken in order to determine the time course of the process by which information derived from a visual target is used to accurately set the amplitude of a simple motor response. We refer to this process as response specification. Separate auditory and visual cues were given to the subjects in order to independently control the moment of response initiation and the time available for processing amplitude information from the target. Six subjects initiated impulses of isometric force in synchrony with the last of predictable series of regular tones. Response amplitudes were to match one of three visual target steps occurring at random times between 0 and 400 ms before the response-synchronizing tone. Using these separate auditory and visual cues, we were able to systematically vary the time interval between target presentation and response onset, termed here Stimulus-Response or S-R interval. Target steps were presented in blocks of either predictable (simple condition) or unpredictable (choice condition) amplitudes. The peak forces and the peaks of their time derivatives were analyzed to determine how subjects achieved accuracy under the different conditions and at different S-R intervals. The trajectories of responses produced in the simple condition were independent of the S-R interval. In contrast, when targets were presented in unpredictable order, the distribution of the peak forces of the subjects' responses depended on the S-R interval. At short S-R intervals (less than 125 ms), subjects made responses whose peak forces were distributed around the center of the range of target steps. These responses formed a unimodal, but broad distribution which was independent of actual target amplitude. With increasing S-R interval (greater than 125 ms), the distributions of peak forces gradually shifted toward the correct target amplitudes, with the means reaching the appropriate amplitudes at S-R intervals of 250-400 ms. At S-R intervals comparable to a reaction time, the range of peak forces was constricted to a similar extent as previously observed in a reaction time task (Hening et al. 1988). We found that the gradual improvement of accuracy was not achieved through changes in trajectory control: at all S-R intervals, subjects utilized a pulse-height control policy (Gordon and Ghez 1987a). Different peak forces were achieved by varying the rate of rise of force, while force rise time was held relatively invariant.(ABSTRACT TRUNCATED AT 400 WORDS)

Development of vertical vestibulo-ocular reflex characteristics in intact and flocculectomized rabbits visually deprived from birth.

The vertical vestibulo-ocular reflex (VOR) was recorded in dark-reared rabbits 3 months of age submitted in the dark to lateral sinusoidal oscillations of different frequencies and fixed amplitude. While the phase of the response was perfectly adequate to ensure head movements compensation, the gain values recorded showed a clear reduction with respect to the values obtained in a normally raised control group of the same age. After exposure to light, the visually deprived animals showed a complete and rapid recovery of normal VOR gain values. Another group of animals was flocculectomized prior to light exposure. The bilateral ablation of flocculus and paraflocculus did not affect the VOR characteristics of the deprived animals. After exposure to light, in the flocculectomized animals, no recovery of the VOR gain values was observed. The present results confirm that visual experience in early life is necessary for a correct development of the VOR. If visual deprivation is limited to the first few months of life, the impairment of the reflex characteristics is completely reversible. Finally our data suggest that in the rabbit, the flocculus controls the early life development of the VOR.

Development of the dynamic characteristics of the horizontal vestibulo-ocular reflex in infancy.

The horizontal vestibulo-ocular reflex (VOR) has been recorded electrooculographically in normal infants aged from one to four weeks and in older subjects submitted to sinusoidal oscillations (+/- 15 degrees, 0.05-0.5 Hz) in the dark. The analysis of the dynamic characteristics of the reflex indicates that, at least in the low frequency range, the VOR has not yet attained a truly compensatory role in the first month of life. In two of the subjects the VOR has been followed throughout the second month of life. At this age the phase of the reflex has already developed the characteristics of the comparison-group at all the frequencies of stimulation, while the trend of the amplitude modulation has not yet changed from the newborn stage. By the 6th month of life the VOR characteristics are comparable with those present in the comparison-group. It appears therefore that the horizontal VOR undergoes a process of maturation during the early period of life, which affects in different times both phase and amplitude values. The possible role exerted by different neural structures on this maturation process is discussed.

Recovery of the vertical vestibulo-ocular reflex gain in rabbits submitted to bilateral and unilateral visual deprivation from birth.

Rabbits were raised in complete darkness from birth to the age of 3 months. At this age, the animals were submitted to dynamic vestibular stimulation consisting of lateral sinusoidal oscillations of different frequencies and fixed amplitude. The vertical VOR, elicited in complete darkness, was then recorded. While the phase of the response was perfectly adequate to ensure head movements compensation, the gain values recorded were clearly reduced with respect to the values obtained in a normally raised control group of the same age. After exposure to light, the visually deprived animals showed a complete recovery of normal VOR gain values in a relatively short period of time. Another group of animals was submitted to monocular prolongation of light deprivation during the fourth month of life. After 2 weeks these rabbits displayed a clear unbalance of the VOR between the two eyes: the eye in which vision was allowed showed a complete recovery of VOR gain values, while the gain of the occluded eye remained unchanged. The present results confirm that visual experience in early life is necessary for a correct development of the VOR. If visual deprivation is limited to the first few months of life, the impairment of the reflex characteristics is completely reversible. Finally, data on monocular deprivation suggest that, in the rabbit, the neural structures which preside to the development of the vertical VOR compensatory properties are lateralized.

EMG responses of the vertical eye muscles to dynamic and static natural vestibular stimulation about different axes in alert rabbits.

The EMG responses of the vertical eye muscles have been recorded in alert intact rabbits submitted to both dynamic and static natural vestibular stimulations about different axes. Following dynamic vestibular stimulation, the phase lead of the EMG response with respect to head position as well as its sensitivity increased with stimulus frequency in all the stimulation modalities. This indicates a progressive recruitment by the stimulus of the second-order vestibular neurons related to semicircular canals. The sensitivity of the response was consistently higher in the effective intermediate roll-pitch modality for all the 4 muscles. Following static stimulation, the EMG response showed an asymmetric modulation. The excitatory response was always higher than the inhibitory one and linearly related with the stimulus. Also for the static stimulation the amplitude of the response was significantly higher when the animal was tilted about the effective intermediate roll-pitch axis for all the 4 muscles. The presence of a maximal EMG response in the same modality for both static and dynamic stimulation indicates a similar spatial organization of those subgroups of ampullar and macular receptors projecting to the same eye muscle.

Vestibular mechanisms related to vertical and torsional eye movements.

1. A series of experiments concerning the organization of the vertical vestibuloocular reflex (VOR) of the rabbit are reviewed. 2. Unanesthetized, encéphale isolé and intact, alert rabbits have been submitted to natural stimulation of the labyrinth consisting in sinusoidal oscillations and static tilts around the longitudinal axis. The oculomotor responses have been recorded at the level of the III nucleus oculomotor neurons (OMN) and from the four vertical eye muscles. 3. The phase and gain of the OMN response at different frequencies of oscillation indicate a serial involvement of the macular ampullar labyrinthine receptors in the activation of the OMN. In the low frequency range of head movements, macular receptors alone can provide adequate ocular compensation. By raising the frequency of head movements, the threshold value of acceleration for recruiting second order vestibular neurons related to semicircular canals is reached and the vertical VOR is controlled predominantly by the ampullar receptors of the vertical canals. 4. These results have been confirmed and extended in the alert, intact rabbit. By orienting the animal in different positions with respect to the axis of oscillation it has been possible to activate selectively one or the other pair of complementary vertical canals. The EMG responses recorded from the vertical eye muscle exhibited different magnitudes in the different orientations, while the phase curve remained the same. 5. The static otolith-ocular reflex has also been investigated by tilting the alert animals oriented in the same way. The pattern of the EMG responses elicited by the pure macular input coincides with that obtained during dynamic stimulation. The existence of separate functional macular regions specifically connected with the different eye muscles and a close convergent action of the two types of labyrinthine receptors in ocular compensation, are suggested.

Phase changes induced by ketamine in the vertical vestibulo-ocular reflex in the rabbit.

The effect of ketamine has been tested on the phase of the vertical vestibulo-ocular reflex of rabbits sinusoidally oscillated at various frequencies. A significant phase lag, predominantly affecting the macular component of the reflex, was observed. This action resembles that induced by Nembutal in the same preparation. A specific action of ketamine on synaptic transmission is suggested. Erroneous phase relationship between natural stimuli responses can be obtained in experiments employing ketamine.