Defining the landscape of ATP-competitive inhibitor resistance residues in protein kinases.

Kinases are involved in disease development and modulation of their activity can be therapeutically beneficial. Drug-resistant mutant kinases are valuable tools in drug discovery efforts, but the prediction of mutants across the kinome is challenging. Here, we generate deep mutational scanning data to identify mutant mammalian kinases that drive resistance to clinically relevant inhibitors. We aggregate these data with subsaturation mutagenesis data and use it to develop, test and validate a framework to prospectively identify residues that mediate kinase activity and drug resistance across the kinome. We validate predicted resistance mutations in CDK4, CDK6, ERK2, EGFR and HER2. Capitalizing on a highly predictable residue, we generate resistance mutations in TBK1, CSNK2A1 and BRAF. Unexpectedly, we uncover a potentially generalizable activation site that mediates drug resistance and confirm its impact in BRAF, EGFR, HER2 and MEK1. We anticipate that the identification of these residues will enable the broad interrogation of the kinome and its inhibitors.

Adrenergic involvement in the locus ceruleus and adjoining regions in the facilitation of predatory attack behavior as induced by hypothalamic stimulation in cats.

The present study was carried out in five cats which did not attack the rats spontaneously. Predatory attack on an anaesthetized rat was elicited by electrical stimulation of extreme lateral regions of hypothalamus. These sites were stimulated at a current strength from 300-700 microa to evoke a predatory attack on an anaesthetized rat. The attack was accompanied by minimal affective display such as alertness, pupillary dilatation, and culminated in beck biting at higher current strength. A scoring system allowed the construction of stimulus response curves, which remained fairly constant when repeated over a period of 3-4 weeks. Microinfusions of norepineprine and clonidine in 4.0 and 5.0 microg dose respectively in locus ceruleus and adjoining tegmental fields facilitated the predatory attack and there was a significant reduction in the threshold current strength for the elicitation of affective and somatomotor components. Microinfusions of yohimbine, an alpha-2 blocker, in 5 microg dose completely blocked the predatory attach response as indicated by an increase in the threshold current strength for the affective components. The somatomotor components were completely inhibited and could not be elicited even when the current strength was increased to 1000 microA. The predatory attack behavior remained completely inhibited for almost two hours following microinfusion of yohimbine. During this period, the animal was extremely drowsy and reacted very slowly even to a painful stimulus such as pinching of tail. Microinfusions of propranalol (beta-blocker), practalol (beta-1 blocker), prazosin (alpha-1 antagonist), propylene glycol as well as saline in similar volumes (0.5 microl) as control failed to produce any blocking effect, thus indicating the involvement of alpha-2 adrenoceptive mechanisms in the modulation of predatory attack in this region of midbrain. The facilitatory effects of norepinephrine and clonidine were significant at P<0.01 and P<0.05 respectively with Wilcoxon's signed rank test. The inhibitory effects of yohimbine were significant at P<0.05. The present study indicates the involvement of alpha-2 adrenoceptive mechanisms in the facilitation of hypothalamically elicited predatory attack.

Enkephalinergic involvement in substantia nigra in the modulation of hypothalamically-induced predatory attack behavior.

The present study was carried out in five cats which did not attack the rats spontaneously. Predatory attack on an anaesthetized rat was elicited by electrical stimulation of lateral hypothalamus at a mean current strength of 650 microA. The attack was accompanied by minimal affective display and culminated in neck biting. Microinfusions of DAME (delta-alanine methionine enkephaline) in 500 ng dose in substantia nigra facilitated the predatory attack and there was a significant reduction in the threshold current strength for affective display as well as somatomotor components. Microinfusions of naloxone, an opioid antagonist in 1.0 microg dose when DAME effect was at its peak reversed the facilitatory effects and the threshold returned to the control levels within 10 minutes of naloxone infusion at the same locus. Microinfusions of naloxone alone in similar dosage completely blocked the predatory attack response as indicated by an increase in the threshold current strength for somatomotor as well as affective display components. The somatomotor were completely inhibited and could not be elicited even when the current strength was increased to 1000 microA. Control injections of saline in similar volumes (0.5 microl) failed to produce any response Microinfusions of naloxone in lower dose (250 ng) failed to produce any blocking effect. These findings indicate that hypothalamically elicited predatory attack is facilitated by enkephalinergic mechanisms operating at the midbrain level.

Hyperalgesic response in rats fed sucrose from weaning to adulthood: role of VMH.

The ventromedial nucleus of hypothalamus (VMH) is implicated in food intake, food preference, nociception and its modulation by palatable food. Palatable drink for 5-48 h in adult rat produced hyperalgesia, which is mediated by VMH. The effect of palatable dietary supplement after weaning on the nociceptive response in adult rats has not been reported. Whether or not VMH influences these nociceptive responses is also not known. The present study was therefore undertaken to investigate the effect of VMH lesion on the nociceptive responses in adult rats ingesting (ad libitum) sucrose from weaning. Weanling rats received sucrose solution in addition to drinking water and laboratory pellets (sucrose-fed group), while the control group of rats received laboratory pellets alone. On attaining adulthood, the behavioral responses, namely tail flick latency (TFL), thresholds of tail flick (TF), vocalization during stimulus (SV), and vocalization after discharge (VA) to phasic and formalin pain rating (FP) to tonic noxious stimuli, were noted in pre- and post-VMH lesion states of both groups of rats. In chronic sucrose-fed rats, the TFL was not affected, the thresholds of TF, SV and VA were significantly decreased (P<.001) and the FP was increased in comparison to the control group, suggesting a hyperalgesic response to chronic sucrose ingestion. After the VMH lesion, in sucrose-fed rats, the thresholds of TF, SV and VA remained unaltered, while the FP was attenuated and TFL decreased. In control rats, VMH lesion produced a hyperalgesic response to both the phasic and tonic noxious stimuli. The data indicate that chronic sucrose feeding and VMH lesion differentially affect the nociceptive responses to the phasic and tonic noxious stimuli. These results suggest that chronic sucrose feeding from weaning to adulthood produces hyperalgesia to both the tonic and phasic noxious stimuli (except TFL), which is probably mediated by VMH.

Neurobiological correlates of panic disorder and agoraphobia.

Panic Disorder and agoraphobia offer considerable diagnostic and management challenges, particularly in general practice. We describe a typical case of panic disorder in a young adult. The recent advances in our understanding of brain functions can be used to explain to a certain extent the biologic basis of panic disorder. A hypothetical model integrating current views on panic disorder and agoraphobia has been proposed. The management principles including the role of cognitive therapy and pharmacotherapy have been discussed.

Reversal of hyperalgesia by transplantation in lateral hypothalamic lesioned rats.

Lateral hypothalamus (LHA) plays a very important role in the modulation of nociceptive behaviour. The stimulation of LHA is known to produce analgesia of both tonic and phasic pain. The present study reports hyperalgesia induced by lateral hypothalamic lesions and the effect of fetal (gestation day 16) hypothalamic transplant on the nociceptive response to phasic thermal noxious stimulation [tail flick latency (TFL)] in LHA lesioned rats. The TFL decreased significantly (12.91 +/- 3.91 sec to 10.51+/- 1.23 sec) following LHA lesion. However, after transplantation, the TFL did not change. This is the first report of a hypothalamic transplant inducing recovery of a nociceptive response.

Ventromedial hypothalamic mediation of sucrose feeding induced pain modulation.

Electrophysiological and behavioural studies suggest a modulatory role of ventromedial nucleus of the hypothalamus (VMH) in nociceptive behaviour. Lesion of the VMH produces hyperalgesia and a greater preference for sucrose solution. Hyperalgesia is also produced by sucrose feeding. To explore specifically the contribution of glucoreceptor neurons of the VMH in the mediation of sucrose-fed hyperalgesia, 2-deoxy-D-glucose (2-DG, antimetabolite of glucose) was slowly albeit continuously infused (1 microl/h for 7 days by microinfusion pumps) into the VMH of adult male rats. Simultaneously, the rats underwent tests for their nociceptive responses in control and sucrose-fed states. The tests for nociception, namely, tail flick latency (TFL), thresholds of tail flick (TF), vocalization during stimulus (SV), vocalization after discharge (VA) were recorded at 0500 h. The tests were repeated after 6, 12, and 48 h in 1 M saline (control group) and 2-DG (experimental group) microinfused rats. Rats were presented with sucrose (20%) solution for 48 h at 0500 h ad libitum in addition to food pellets and tap water. Infusion of 2-DG per se in the VMH led to hypoalgesia (in threshold of TF, SV, VA) while feeding sucrose for 6-12 h per se led to hyperalgesia (in TFL, threshold of SV and VA). Sucrose feeding to 2-DG rats, however, attenuated the hypoalgesia of 2-DG as well as the hyperalgesia of sucrose feeding. The results suggest that the VMH glucoreceptor neurons probably modulate sucrose mediated phasic pain responses.

Effect of VMH lesion on sucrose-Fed nociceptive responses.

An initial analgesia followed by hyperalgesia to phasic noxious stimuli occurs after ingestion of sucrose ad libitum. However, the mechanism underlying hyperalgesia is not known. The present study was designed to explore the role of VMH in the mediation of the hyperalgesic effect of sucrose ingestion. Adult male albino rats received sucrose solution (20% p.o.) in addition to laboratory food pellets and tap water ad libitum. Their behavioural responses to various phasic and tonic noxious stimuli were recorded after 6, 12 and 48 h during pre and post-sucrose fed states in both the control and VMH lesion groups of rats. Sucrose feeding to control rats significantly reduced the tail flick latency (TFL) and threshold of vocalization during stimulus (SV) and after discharge (VA) indicating hyperalgesia, while the threshold of tail flick remained unaffected. The average pain rating during the formalin test (tonic pain) decreased significantly indicating analgesia. VMH lesion decreased the latency (mean +/- SD) for tail flick (11.26 +/- 4.65 from 15.61 +/- 5.12 s), threshold (median) for tail flick (0.04 from 0.08 mA), vocalization during stimulus (0.05 from 0.1 mA) and vocalization after discharge (0.15 from 0.2 mA), while the tonic pain rating increased, thereby suggesting a hyperalgesic state. However, sucrose feeding to lesioned rats neither potentiated nor attenuated their hyperalgesia. The results suggest that sucrose feeding for 6-48 h ad libitum produces hyperalgesia to phasic noxious and analgesia to tonic noxious stimuli, while VMH lesion produces hyperalgesia to both phasic and tonic noxious stimuli. Secondly, sucrose ingestion by VMH lesion rats does not affect their responses to pain, suggesting the possible role of VMH in the mediation of sucrose-fed nociceptive responses.

Amygdalar tissue transplants improve recovery of nociceptive behaviour in rats.

PURPOSE: Amygdala plays a very important role in the mediation of emotional and affective components of pain. Bilateral amygdalectomy increases the threshold for vocalization, a measure of emotional reactivity, without any change in the tail flick latency. The present work was designed to study the recovery of emotional nociceptive behaviour following neural tissue transplantation in lesioned rats. METHODS: In a group of adult wistar rats lesions of the central nucleus of amygdala (CeA) were produced electrolytically. In a separate group of rat's amygdalar tissue was transplanted at the lesioned site 2 days thereafter. The vocalization tests, viz. simple vocalization (SV) and vocalization after discharge (VA), which were used to study the emotional nociceptive behaviour were carried out both before and after pro-ducing lesion/transplant. RESULTS: Bilateral CeA lesions increased the thresholds for SV and VA significantly (p < 0.001), indicating analgesia. Following amygdalar tissue transplantation a significant decrease (p < 0.05) in the thresholds were observed when compared with the lesioned group, although when compared with the basal data it showed an increase (p<0.05). CONCLUSIONS: The results indicate a partial recovery of the vocalization response following amygdalar tissue transplantation.

Effect of tonic pain on schedule specific feeding behaviour.

Food deprivation produces analgesia. This response is reversed i.e. pain sensitivity is lowered, when the food deprived rats are fed. In the present study the effect of chronic pain on the motivation to get food, in food deprived rats, was observed. In ten rats the effect of formalin and morphine plus formalin on the motivation to get food was studied. Injection of formalin significantly (P < 0.01) decreased the number of lever presses from 450 +/- 30 to 225 +/- 25. However, after injecting morphine the effect was reversed. The present study shows reduced internal drive to procure food by the food deprived animals, when they were under chronic pain. The effect was blocked by morphine, suggesting the role of opioids in modulating the motivation for getting food.

Neural tissue transplant in the lateral hypothalamic lesioned rats: functional recovery pattern.

Transplantation has come of age as an important investigative tool for studying normal growth and development of the brain tissue. The present study reports the effect of lateral hypothalamic (LHA) lesion and foetal hypothalamic tissue transplantation on the feeding behaviour. In a group of rats, LHA was lesioned bilaterally by passing direct current. Subsequently, in a separate group of rats, foetal hypothalamic tissue was transplanted at the lesioned site. Following LHA lesion, all the rats died of aphagia and adipsia within 7 days, whereas, the rats in whom foetal hypothalamus was transplanted, started taking food and water in small quantities from the first day of transplantation itself. Later, they were able to attain their preoperative values. This recovery of the feeding behaviour following foetal tissue transplantation may be due to the formation of synaptic connections or due to the release of neurotrophic factors.

Challenges of teaching physiology in a PBL school.

A problem-based learning (PBL) curriculum was introduced at McMaster University more than three decades ago. Not many schools have adopted the system despite its distinct advantages. The present paper examines the challenges of teaching physiology in a PBL curriculum and gleans through the literature supporting PBL. It appears that one of the reasons why PBL is not becoming readily acceptable is the lack of concrete reports evaluating the curricular outcomes. The suggestion (R.E. Thomas. Med Educ. 31:320-329, 1997) to standardize and internationalize all components of validated PBL curricula is quite valid. A database needs to be generated that can be easily accessed by traditional institutions to see the rationality and easy implementation of the PBL curriculum.

Neuronal responses of periaqueductal gray to peripheral noxious stimulation.

Central pathways transmit pain from peripheral regions to one of the most important area of the descending pain modulatory system, the Periaqueductal gray (PAG). Independent discoveries in the past suggest that the PAG contains afferent input, output neurons and intrinsic interneurons. An attempt was made in the present study to find out the effects of more than one kind of noxious stimulus on the PAG neuronal activity. Experiments were conducted in rhesus monkeys and the effects of noxious mechanical, thermal and tooth-pulp stimulation on the activity of 14 neurons were studied. The neurons responded to more than one kind of noxious stimuli by increasing or decreasing its firing rate. No limb specificity could be identified and homogeneous distribution of the excitatory and inhibitory neurons was found.

Hypothalamo-limbic involvement in modulation of tooth-pump stimulation evoked nociceptive response in rats.

The hypothalamo-limbic system has been implicated in recognizing the affective significance of pain and elicitation of related emotional responses. Several evidences from different studies support a role of these areas in endogenous analgesic mechanisms for pain modulation as elucidated by different pain tests in more than one animal model. In the above context, the aim of this study was to investigate the relative effectiveness of the pain modulatory action of hypothalamic and limbic structures in rat using similar stimulation parameters, and studying the effect on tooth pulp stimulation evoked jaw opening reflex (TP-JOR). To achieve the objective, unilateral stimulation of hypothalamic (lateral = LH; ventromedial = VMN; anterior = AH) and limbic areas (amygdala = AMYG; hippocampus = HIPP) was done on the TP-JOR test. A significant reduction in the amplitude of EMG recorded from the digastric muscle (dEMG) as a result of tooth-pulp stimulation was observed on stimulation of LH, VMN, AMYG and HIPP but not from AH. Also, the magnitude of this effect was almost similar from these areas. The results suggest that these areas (except AH) have an antinociceptive role in tooth-pulp stimulation evoked pain response.

Enkephalinergic mechanisms in midbrain (dPAG) in modulation of hypothalamically-induced predatory attack behaviour.

Present study was carried out in nine cats which did not attack the rats spontaneously. Predatory attack on an anaesthetized rat was elicited by electrical stimulation of lateral hypothalamus at a mean current strength of 690 microA. The attack was accompanied by minimal affective display and culminated in neck biting. Microinjections of delta-alanine methionine enkephaline (DAME) in 250 ng dose in dorsal periaqueductal gray completely suppressed the predatory attack. There was a significant increase in the threshold current strength for affective display components while the somatic components were completely inhibited even when the current strength was increased to 1000 microA. Microinjections of naloxone, an opioid antagonist in 1 microgram dose reversed the DAME blocking effect and the thresholds returned to control levels within 10 min of microinjections. Microinjections of naloxone alone in similar dose facilitated the response as indicated by a decrease in threshold current strengths for both affective display and somatomotor components. Control injections of saline in similar volumes (0.5 microliter) failed to produce any change. These findings indicate that hypothalamically induced predatory attack is inhibited by enkephalinergic mechanisms operating at the dPAG level in the midbrain.

Role of midbrain ventro-lateral tegmental area (VTA) adrenergic mechanisms in facilitation of hypothalamically-induced predatory attack behaviour.

Bipolar concentric electrodes were implanted in extreme lateral regions of hypothalamus having coordinates (A12.5 mm, L 3.5-3.7 mm, V 3.0-3.7 mm). These sites were electrically stimulated using biphasic square wave pulses (1 ms, 60 Hz) at a current strength ranging from 300-800 microA to evoke predatory attack on an anaesthetized rat. At lower current strengths of 300 microA only altertness with pupillary dilatation was produced. Gradual increase in the current strength led to recruitment of somatic and affective components and a full blown predatory attack on a rat was produced at a mean current strength of 700 microA. A scoring system allowed the construction of stimulus response curve, which remained fairly constant when repeated over a period of 3-4 weeks. In ventrolateral tegmental area (VTA), bilateral microinjections of norepinephrine (NE 10 micrograms in 0.5 microliter saline, pH 7.4) lowered the mean threshold current strength to 100 microA while predatory attack was produced at 500 microA. Clonidine (5 micrograms in 0.5 microliter propylene glycol, pH 7.4) an alpha-2 agonist similarly lowered the mean threshold to 100 microA and predatory attack threshold to 400 microA. The effects of clonidine appeared within 20 min of microinjection and persisted up to 6 hr. Yohimbine, an alpha-2 antagonist (4 micrograms in 0.5 microliter propylene glycol. pH 7.4) when microinjected into the same locus (VTA), completely blocked predatory attack behaviour for 3 days, the peak period of the blocking effects were between 3-8 hr, after microinjection. Isoproterenol (beta agonist), propranolol (beta blocker), prazosin (alpha-1 antagonist) and phenoxybenzamine (alpha antagonist) failed to produce any effect. Normal saline and propylene glycol in similar volumes served as controls. The excitatory effects of NE and clonidine and inhibitory effects of Yohimbine were significant at P < 0.01 and P < 0.05 respectively with Wilcoxon's signed rank test. The present study indicates the involvement of alpha-2 adrenoceptive mechanisms operating at the midbrain (VTA) level in the elicitation of predatory attack from lateral hypothalamus.

Role of midbrain ventro-lateral tegmental area (VTA) enkephalinergic mechanisms in the facilitation of hypothalamically-induced predatory attack behaviour.

Bipolar concentric electrodes were implanted in five cats in extreme lateral regions of hypothalamus. These sites were electrically stimulated using biphasic square wave pulses at a current strength ranging from 300-800 microA to evoke predatory attack on an anaesthetized but live rat. At lower current strength (300 microA) only alertness with pupillary dilatation was produced. Gradual increase in the current strength led to the recruitment of somatic and affective components and a predatory attack was exhibited at a mean current strength of 700 microA. A scoring system allowed the construction of stimulus response curves, which remained fairly constant when repeated over a period of 3-4 weeks. Bilateral microinjections of delta-alanine methoinine enkephaline (DAME) (500 ng in 0.5 microliter saline) in ventrolateral tegmental area (VTA) elevated the mean threshold current strength for affective components while somatomotor components were totally inhibited. The blocking effect of DAME persisted for 1 hour. Microinjections of naloxone (1 microgram) in similar volumes facilitated the response as indicated by a reduction in threshold current strength for somatomotor and affective components. Microinjections of naloxone (1 microgram) in similar volumes facilitated the response as indicated by a reduction in threshold current strength for somatomotor and affective components. Microinjections of naloxone (1 microgram) also reversed the blocking effect of DAME and the thresholds returned to the control level within 10 min while microinjection of normal saline as control had no effect. The excitatory effects of naloxone and inhibitory effects of DAME were statistically significant at P < 0.01 and P < 0.05 respectively with Wilcoxon's signed rank test. The present study indicates that enkephalinergic as well as opioidergic mechanisms operating at the midbrain (VTA) level are involved in the inhibition of predatory attack as elicited from lateral hypothalamus.

Influence of low and high doses of naloxone on tonic pain and motor activity in rats.

Naloxone has been reported to affect pain and locomotor activity differently depending on the dose. The objective of the present investigation was to study the effects of low and high (6 micrograms and 3 mg/kg, s.c.) doses of naloxone (Nx) on formalin-induced pain (tonic pain) and spontaneous motor activity and any correlation between them. The experiments were conducted on adult male Wistar rats. Tonic pain and spontaneous motor activity were recorded by the formalin test and video monitoring respectively. An increase in spontaneous motor activity (locomotion, movements and distance) was observed following formalin injection as compared to basal activity (P < 0.05). Low dose of Nx reduced the pain intensity and also the spontaneous motor activity during the later phase (after 15 min of formalin injection) (P < 0.05). High dose of Nx on the other hand increased the pain intensity but still reduced motor activity (P < 0.05). Both doses of Nx initially produced hyperalgesia (5 min peak). The bidirectional effects of Nx on formalin pain were dissociated from the spontaneous motor behavior of rats. A direct correlation could not be established between pain intensity and spontaneous motor activity.

2-buten 4 olide affects feeding behavior of rhesus monkey.

1. Endogenous satiety substance 2-Buten 4 Olide (2-B40- a short chain sugar derivative) effect on feeding behaviour of rhesus monkey was studied. 2. The cannula was implanted in the third ventricle of three, adult, male, rhesus monkeys. 3. Monkeys were conditioned to ingest their daily requirement of food during one hour. 4. Their daily food intake was recorded. 5. Various doses of 2-B40 (1.6, 2.1, 2.7, 4.3, 10.6, 20 and 25 mg)were administered intracerebroventricularly on different days five minutes prior to presentation of food. 6. The effective dose for inducing significant satiety effect was 20.0 mg, while 10.6 mg induced a mild and 25.0 mg a severe effect. 7.However, when administered intraperitoneally 43 mg/kg bw 2-B40 induced satiety effect, but was ineffective in doses of 1.5 to 21.5 mg bw. 8. This study is suggestive of the presence of 2-B40 satiety mechanism in monkeys almost in doses similar to those previously reported for rats.