Usefulness of 18F-α-Methyltyrosine PET for Therapeutic Monitoring of Patients with Advanced Lung Cancer.

BACKGROUND/AIM: L-[3-18F]-α-methyl tyrosine (18F-FAMT) positron emission tomography (PET) has a high specificity for detecting malignant lesions. However, the usefulness of therapeutic monitoring of 18F-FAMT PET against advanced human neoplasms remains unclear. Here, we evaluated 18F-FAMT PET clinical significance regarding therapy response and outcome after systemic chemotherapy in patients with advanced lung cancer, compared to 18F-FDG PET. PATIENTS AND METHODS: All patients with untreated advanced lung cancer received 18F-FAMT PET and 18F-FDG PET before and 4 weeks after one cycle of chemotherapy. Metabolic response (MR) was defined according to the PERCIST guideline. RESULTS: Ninety-five patients were eligible for analysis on both PET scans. The histological type included 87 non-small cell lung cancers and 8 small-cell lung cancers. Post-treatment maximal standardized uptake values (SUVmax) and MR on 18F-FAMT PET were correlated with tumor response. In all patients, post-treatment SUVmax of 18F-FDG and 18F-FAMT PET and MR of 18F-FAMT PET were statistically significant prognostic markers for predicting poor outcome by univariate analysis. Multivariate analysis confirmed that MR on 18F-FAMT PET was a significant independent prognostic factor. CONCLUSION: MR on 18F-FAMT PET may be a potential parameter to predict the prognosis after first-line chemotherapy in patients with advanced lung cancer.

Effects of the dopamine stabilizers (S)-(-)-OSU6162 and ACR16 on prolactin secretion in drug-naive and monoamine-depleted rats.

Dopaminergic stabilizers may be conceptualized as drugs with normalizing effects on dopamine-mediated behaviours and neurochemical events. (S)-(-)-OSU6162 (OSU6162) and ACR16 are two structurally related compounds ascribed such properties, principally because of their stabilizing effects on motor activity in rodents. Reports in the literature indicate possible partial D2 receptor agonist effects using various in vitro systems. This study aimed to measure D2 receptor antagonist and agonist effects of OSU6162 and ACR16 in vivo. To address this, we have studied the effects of both compounds on prolactin secretion in drug-naive and dopamine-depleted rats; dopamine depletion was induced by pretreatment with reserpine plus α-methyl-DL: -p-tyrosine. We find that OSU6162 and ACR16 both stimulate prolactin secretion in drug-naive rats with OSU6162 being considerably more potent and efficacious. Both compounds show a non-significant trend towards reversal of the increased secretion caused by dopamine depletion, whereas the D2 receptor antagonist haloperidol further increased prolactin secretion. Thus, this study suggests that OSU6162 and ACR16 act as D2 receptor antagonists under normal conditions in vivo, possibly with minor agonist effects in a state of dopamine depletion.

Conditional mouse mutants highlight mechanisms of corticotropin-releasing hormone effects on stress-coping behavior.

Hypersecretion of central corticotropin-releasing hormone (CRH) has been implicated in the pathophysiology of affective disorders. Both, basic and clinical studies suggested that disrupting CRH signaling through CRH type 1 receptors (CRH-R1) can ameliorate stress-related clinical conditions. To study the effects of CRH-R1 blockade upon CRH-elicited behavioral and neurochemical changes we created different mouse lines overexpressing CRH in distinct spatially restricted patterns. CRH overexpression in the entire central nervous system, but not when overexpressed in specific forebrain regions, resulted in stress-induced hypersecretion of stress hormones and increased active stress-coping behavior reflected by reduced immobility in the forced swim test and tail suspension test. These changes were related to acute effects of overexpressed CRH as they were normalized by CRH-R1 antagonist treatment and recapitulated the effect of stress-induced activation of the endogenous CRH system. Moreover, we identified enhanced noradrenergic activity as potential molecular mechanism underlying increased active stress-coping behavior observed in these animals. Thus, these transgenic mouse lines may serve as animal models for stress-elicited pathologies and treatments that target the central CRH system.

Intra-individual comparison of p-[123I]-iodo-L-phenylalanine and L-3-[123I]-iodo-alpha-methyl-tyrosine for SPECT imaging of gliomas.

OBJECTIVES: Radioactive amino-acids accumulate in gliomas even with an intact blood-brain-barrier. L-3-[(123)I]-iodo-alpha-methyl-tyrosine (IMT) is well established for SPECT imaging of gliomas. Recently, we introduced p-[(123)I]-iodo-L-phenylalanine (IPA) for the characterisation of brain lesions. This study compares both tracers in glioma patients. METHODS: Eleven patients with gliomas (1 WHO grade 1, 5 grade 2, 1 grade 3, 2 grade 4 gliomas, 1 unconfirmed upgrading and 1 post-therapeutic non-neoplastic lesion) underwent SPECT imaging with IPA (early and delayed acquisitions at 30 min and 3 h) and IMT (early only). Maximum tumour-to-brain ratios (TBR) were calculated using region-of-interest analysis to assess uptake of IMT and IPA. Imaging results were compared to histopathological findings. RESULTS: Early TBRs of IMT and IPA were strongly correlated (r = 0.828, p = 0.002). TBRs were higher for IMT than IPA (1.95+/-0.50 versus 1.79+/-0.42; p < 0.05), but independent from tumour cell density (p > 0.1). Visual interpretation by different observers was more concordant for IMT-SPECT than IPA-SPECT (kappa 1.0 versus 0.774). No differences in early TBRs were observed between low-grade and high-grade gliomas for IMT (1.97+/-0.53 versus 2.21+/-0.44, p > 0.5) or IPA (1.70+/-0.23 versus 2.21+/-0.56, p = 0.167) with a trend to higher TBRs in low-grade tumours for IMT (p = 0.093). In contrast to the known wash-out of IMT, we observed persistent accumulation of IPA in gliomas. CONCLUSIONS: IPA shows lower TBRs than IMT, especially in low-grade tumours, so IMT should be preferred for the delineation of low-grade gliomas by SPECT imaging. Due to its prolonged retention, however, IPA remains promising for therapeutic use in gliomas after labelling with I-131.

Splenic norepinephrine depletion following acute stress suppresses in vivo antibody response.

Exposure to an intense acute stressor immediately following immunization leads to a reduction in anti-KLH IgM, IgG, and IgG2a, but not IgG1. Stress also depletes splenic norepinephrine (NE) content. Immunization during pharmacological (alpha-methyl-p-tyrosine) or stress-induced splenic NE depletion results in antibody suppression similar to that found in rats immunized prior to stressor exposure. Prevention of splenic NE depletion during stress by tyrosine, but not pharmacological elevation (mirtazapine) of NE, resulted in normal antibody responses. These data support the hypothesis that splenic NE depletion is necessary and sufficient for stress-induced suppression of antibody to a T-cell dependent antigen.

The partial D2-like dopamine receptor agonist terguride acts as a functional antagonist in states of high and low dopaminergic tone: evidence from preweanling rats.

RATIONALE: In adult rats, the partial D(2)-like agonist terguride acts as an antagonist at normosensitive D(2)-like post-synaptic receptors, while it acts as an agonist at the same receptors during states of low dopaminergic tone. OBJECTIVE: The purpose of the present study was to determine whether partial D(2)-like agonists exhibit both antagonistic and agonistic actions during the preweanling period. METHODS: In experiments 1 and 2 (examining the agonistic actions of terguride), preweanling rats were either given an escalating regimen of amphetamine to induce a state of amphetamine withdrawal or pretreated with the tyrosine hydroxylase inhibitor AMPT. Distance traveled was measured after rats were injected with saline, terguride (0.4-1.6 mg/kg), or the full D(2)-like receptor agonist NPA (0.01 mg/kg). In experiment 3 (examining the antagonistic actions of terguride), preweanling rats were pretreated with terguride 30 min before they were tested with saline, NPA (0.05 mg/kg), or amphetamine (1.5 mg/kg). RESULTS: NPA had an exaggerated locomotor activating effect when tested under conditions of amphetamine withdrawal, while the partial D(2)-like agonist did not enhance distance traveled under any circumstance. Similarly, NPA increased and terguride did not affect the distance-traveled scores of AMPT-pretreated rats. In experiment 3, terguride pretreatment significantly reduced the distance traveled of amphetamine-treated and NPA-treated rats. CONCLUSIONS: The behavioral evidence indicates that, during the preweanling period, terguride antagonizes D(2)-like post-synaptic receptors in a state of high dopaminergic tone; however, there is no evidence that terguride is capable of stimulating D(2)-like post-synaptic receptors during states of low dopaminergic tone.

Evidence in locomotion test for the functional heterogeneity of ORL-1 receptors.

1. The ORL1 agonists nociceptin and Ro 64-6198 were compared in their ability to modify spontaneous locomotor activity in male NMRI mice not habituated to the test environment. 2. Higher doses of nociceptin (>5 nmol i.c.v.) reduced whereas lower doses (<1 nmol i.c.v.) stimulated locomotor activity. Both effects were blocked by the putative ORL1 antagonists [NPhe1]nociceptin(1-13)NH2 (10 nmol i.c.v.) and UFP101 (10 nmol, i.c.v.). The effects were also blocked by naloxone benzoylhydrazone (1 mg x kg(-1) s.c.), but not by the nonselective opioid antagonist naloxone (1 mg x kg(-1) s.c.). 3 In contrast to nociceptin, the synthetic ORL1 agonist Ro 64-6198 (0.01-1.0 mg x kg(-1) i.p.) produced monophasic inhibition of locomotor activity, which was insensitive to the treatment with [NPhe1]nociceptin(1-13)NH2 or naloxone benzoylhydrazone. Treatment with UFP101 abolished the locomotor inhibition induced by Ro 64-6198 (1.0 mg x kg(-1)), whereas naloxone (1.0 mg x kg(-1), s.c.) further increased the locomotor-inhibitory effects. 4. Naloxone benzoylhydrazone (0.3; 1.0 and 3.0 mg x kg(-1) s.c.) increased locomotor activity, although the effect was statistically significant only with the highest dose used. 5. Pretreatment with the tyrosine hydroxylase inhibitor H44-68 totally eliminated the motor-stimulatory effects of low doses of nociceptin, probably via dopamine depletion. 6. The results suggest that nociceptin stimulates locomotor activity at low doses if dopamine activity is intact. High doses of nociceptin and all the tested doses of Ro 64-6198 seem to interact with a functionally different subset of ORL1 receptors. In addition, the effects of Ro 64-6198 are modulated by tonic opioid receptor activity.

Effect of p-chlorophenylalanine and alpha-methyltyrosine on the antinociceptive effect of antidepressant drugs.

The role of para-chlorophenylalanine and alpha-methyl-DL-p-tyrosine in the antinociceptive effects of the intracerebroventricular administration of the antidepressant drugs clomipramine, zimelidine, imipramine and maprotiline was studied using the acetic acid writhing test in mice. The results demonstrated an antinociceptive effect for all these antidepressants. Pretreatment with para-chlorophenylalanine significantly reduced the antinociception induced by the ED50's of imipramine and maprotiline, and did not modify the effects of zimelidine and clomipramine, pretreatment with alpha-methyl-tyrosine did not modify the antinociception induced by these drugs except maprotiline. Pretreatment with para-chlorophenylalanine plus alpha-methyltyrosine significantly reduced the antinociceptive effect of all the antidepressants tested. The main finding of the present study is that the association of para-chlorophenylalanine plus alpha-methyltyrosine reduced the antinociceptive action of all the antidepressants. This means that critical levels of both 5-HT and NA are responsible for mediating the antinociceptive effects of antidepressants on the writhing test in mice.

The role of alpha-adrenergic mechanisms within the area postrema in dopamine-induced emesis.

Intracerebroventricular injection of dopamine (0.5-4.0 mg) produced dose-dependent and short-lasting emesis (1-8 min) in cats, which was abolished after ablation of the area postrema. Relatively selective alpha 2-adrenoceptor antagonists (yohimbine and idazoxan) and a mixed alpha 1- and alpha 2-adrenoceptor antagonist (tolazoline), but not a non-selective alpha 1-adrenoceptor antagonist (prazosin), injected intracerebroventricularly inhibited the emesis induced by intracerebroventricular dopamine. However, dopamine receptor antagonists (chlorpromazine, droperidol, spiperone, domperidone, triflupromazine, sulpiride and metoclopramide), an antimuscarinic drug (atropine), a ganglionic blocking agent (mecamylamine), an opioid receptor antagonist (naloxone) and a 5-HT receptor antagonist (methysergide), all injected intracerebroventricularly, had no significant effect on emesis evoked by intracerebroventricular dopamine. The emetic response to intracerebroventricular dopamine was attenuated in cats pretreated with intracerebroventricular reserpine, 6-hydroxydopamine, alpha-methyl-p-tyrosine and hemicholinium-3. It is postulated that dopamine-induced emesis is mediated through the release of noradrenaline acting at alpha 2-adrenoceptors and that it depends on the integrity of monoaminergic and possibly cholinergic structures within the area postrema. It appears, therefore, that the emetic effect of intracerebroventricular dopamine is mediated by adrenergic rather than dopaminergic mechanisms in the area postrema, at least in the cat.

Comparative effects of two dithiocarbamates disulfiram and thiram, on adrenal catecholamine content and on plasma dopamine-beta-hydroxylase activity.

Both disulfiram (tetraethylthiuram disulfide), an alcohol aversive drug, and thiram (tetramethyl-thiuram disulfide), a widely used pesticide, significantly increased the dopamine pool in the adrenal glands of dosed rats. The dopamine increase was detectable within 4 h of oral dosing with 100 mg/kg of either dithiocarbamate and peaked 24 h later at 10 times control values. In control rats the dopamine turnover was 0.51 h-1 as calculated by the assumed first order decline of dopamine after a single injection of alpha-methyl-p-tyrosine (alpha-MT, 400 mg/kg i.p.) resulting in a dopamine-beta-hydroxylase (DBH) activity of 0.73 nmol/h per pair of adrenals. In the adrenals of rats pretreated with thiram and then injected with alpha-MT, the adrenal dopamine content did not significantly decline, indicating that thiram reduced the conversion of dopamine to noradrenaline, eventually leading to the observed dopamine increase. Plasma DBH activity was significantly reduced 4 h and 24 h after dosing with thiram, but was unchanged after treatment with disulfiram. The determination of plasma DBH activity could be a marker to monitor the effect of thiram on catecholamine metabolism in occupationally exposed workers but not that of disulfiram in abstinent alcoholics.

The involvement of noradrenaline, 5-hydroxytryptamine and acetylcholine in imipramine-induced seizures in mice.

The influence of some noradrenergic, 5-hydroxytryptaminergic and cholinergic agents on imipramine-induced seizures were investigated in mice. DL-threo-3,4-dihydroxyphenylserine (DOPS) and pargyline significantly potentiated imipramine-induced seizures. Phentolamine and prazosin significantly attenuated seizures elicited by imipramine and significantly attenuated the seizure-enhancing effect of DOPs. alpha-Methyl-p-tyrosine and reserpine significantly attenuated seizures induced by imipramine. Disulfiram significantly protected mice against imipramine-induced seizures. However, DOPS significantly potentiated seizures induced by imipramine in disulfiram-pretreated animals. Clonidine effectively protected mice against imipramine-induced seizures. Idazoxan, on the other hand, significantly potentiated seizures induced by imipramine and significantly antagonised the protective effect of clonidine against the seizures. 5-HTP, PCPA, cyproheptadine, mianserin, ketanserin and trazodone did not affect imipramine-induced seizures to any significant extent. Physostigmine antagonised seizures induced by imipramine while atropine significantly potentiated the seizures, and significantly attenuated the protective effect of physostigmine against the seizures. These data suggest that enhancement and attenuation of central noradrenergic and cholinergic neurotransmissions respectively, and not 5-HT mechanisms, may underlie imipramine-induced seizures in mice.

Infusion of dopamine at low concentrations stimulates the release of prolactin from alpha-methyl-p-tyrosine-treated rats.

Prolactin (PRL) secretion from the anterior pituitary gland is inhibited by dopamine (DA) released into the hypophysial portal vasculature from neurons in the hypothalamus. We have shown previously that DA also stimulates PRL secretion in vitro. Here we report that DA has a dual effect on PRL release in vivo. Injection of rats with alpha-methyl-p-tyrosine (200 mg/kg, ip) induced an immediate 35-fold enhancement of PRL secretion which reached a plateau by 90 min after injection on diestrus 1. When DA was infused intravenously at varying doses beginning at 90 min after alpha-methyl-p-tyrosine, differing effects on PRL secretion were observed. These effects were dose dependent: higher doses of DA (1000 ng/kg/min) inhibited and lower doses (10 ng/kg/min) stimulated PRL secretion. These data suggest that DA may be an important stimulator of PRL secretion in vivo.

Pressure reversed extracellular striatal dopamine decrease produced by D1 receptor agonist SKF 38393, and D2 receptor agonist LY 171555, but failed to change the effect of the activation of both D1 and D2 receptors.

When human divers or experimental animals are exposed to high pressure, they develop the high-pressure neurological syndrome which is characterized by electroencephalographic changes, and behavioral disturbances. Recently, neurochemical disorders such as a pressure-induced increase in dopamine release have been demonstrated. In the present study, pharmacological experiments, using dopamine receptor agonists such as D1 receptor agonist SKF 38393, D2 receptor agonist LY 171555, and D1/D2 receptor agonist apomorphine, were performed to investigate dopamine receptor function at the neurochemical level. Only apomorphine and mixed SKF 38393 + LY 171555 prevented the pressure-induced increase in dopamine release while SKF 38393 or LY 171555 administered alone failed to do so. The results suggest that the D1-D2 link would be reduced under high pressure because of an abnormal function of D1 receptors which would allow high-affinity D2 states for dopamine. If so, such a preponderance of high-affinity states in D2 postsynaptic receptors could be associated with hyperbaric hyperlocomotor activity. Elsewhere, results also suggested that the pressure-induced disorders in dopamine receptor function could be involved in the pressure-induced elevation in dopamine release.

Alpha-methyltyrosine blocks the expression of rotation classically conditioned with apomorphine.

Rats with unilateral 6-hydroxydopamine lesions of substantia nigra rotate (circle) when placed, undrugged, in the environment in which they have previously been treated with apomorphine. This conditioned rotation, like the unconditioned rotation which acutely follows the administration of apomorphine, is directed away from the side with the lesion, i.e., the rotation is contralateral. Here, rats that had been administered apomorphine weeks earlier were tested, in a crossover design, for the expression of conditioned rotation following treatment with saline and with alpha-methyltyrosine. When administered four hours prior to testing, 100 mg/kg alpha-methyltyrosine significantly antagonized the expression of classically conditioned rotation. In a second group of animals, alpha-methyltyrosine had no effect on the unconditioned rotation induced by 0.05 mg/kg apomorphine.

Alpha-MPT does not affect dopamine levels in the periventricular organ of lizards.

Recent studies indicate that the liquor (CSF)-contacting cell bodies in the periventricular organ of non-mammalian vertebrates accumulates rather than synthesizes dopamine. We therefore gave the lizard Gekko gecko injections of the dopamine synthesis inhibitor alpha-methylparatyrosine (alpha-MPT). Brains were fixed 105-240 min after injection. Subsequent staining with dopamine antiserum revealed almost no changes in staining in the periventricular organ but there was a dramatic decrease in the other dopaminergic cell groups of the brain. The data support the notion that the cells in the periventricular organ accumulate dopamine and that the CSF plays an important role in dopamine neurotransmission in non-mammalian vertebrates.

Long-term depression in striatal dopamine release monitored by in vivo voltammetry in free moving rats.

It was proposed to monitor in free moving rats, by in vivo voltammetry, the effects of intracerebroventricular (i.c.v.) administration of drugs known to act on the synthesis of dopamine (DA), using an original multifiber carbon electrode which enables without-discontinuity long-term recordings in extracellular DA release. Results show that i.c.v. administration of alpha-methyl-p-tyrosine, gamma-butyrolactone, and apomorphine induced long-term depression in striatal DA release, over periods of time of more than 24 h. These results are in agreement with the dopaminergic hypothesis; and we conclude that i.c.v. administration of drugs and the use of the multifiber carbon electrode constitutes a valuable tool to monitor DA metabolism in chronically implanted animals.

Neurotoxic lesion of the mesencephalic reticular formation and/or the posterior hypothalamus does not alter waking in the cat.

In order to re-evaluate the role of two putative waking systems, we injected a neural cell body toxin, ibotenic acid (IA) (45 micrograms/microliters), into the mesencephalic reticular formation (MRF) and/or the posterior hypothalamus (PH). On the one hand, when the cell body destruction was only restricted to the MRF, the IA microinjection was followed by a temporary high voltage and slow neocortical electroencephalogram (EEG) during the first 24 postoperative hours and by a subsequent long term increase in waking which lasted 8-12 h. After the first postoperative day, there were no motor disturbances, no aphagia nor adypsia, no alteration of cortical activation and no modification of thermoregulation or of the sleep-waking cycle. On the other hand, the IA microinjection into the PH induced a hypothermia during the first postoperative night and a dramatic transient hypersomnia immediately after the disappearance of the anesthesia (14-24 h after the IA injection). On the third day, all cats recovered control level of paradoxical sleep (PS), slow wave sleep (SWS) and cerebral temperature. They presented normal motor behavior but they were not able to eat by themselves during the first postoperative week. Finally, when the lesions of the MRF and the PH were realized in one single operation, the cats were first motionless in a comatose state for 2-3 days. This state was accompanied by a transitory hypothermia and the suppression of a spontaneous or evoked cortical low voltage fast activity. However, from the 2nd postoperative week, both behavioral and EEG waking re-occurred. By contrast, the two successive operations (MRF followed by PH) did not induce a comatose state. We did not observe any deficit in motor behavior, and the sleep-waking cycle was quite normal as from the second postlesion day. In the MRF-PH-lesioned cats, the injection of alpha-methyl-p-tyrosine (150 mg/kg) induced a large decrease in waking and a moderate increase in PS. In the MRF-lesioned cats, IA produced a large area of cell body loss, centered in the MRF, that extended from levels A2 to A6 of stereotaxic planes and sometimes encroached upon the red nucleus and the substantia nigra. In the PH-lesioned cats, the histological analysis revealed a great loss of cell bodies in the PH extended from levels A8 to A12.5. The damage included the lateral and posterior hypothalamic areas and sometimes the tuberomamillary nucleus. In MRF- and PH-lesioned cats, the cell body loss extended from levels A2 to A12.5.(ABSTRACT TRUNCATED AT 400 WORDS)

Presynaptic effects of melatonin on norepinephrine release and uptake in rat pineal gland.

The effect of melatonin injection on norepinephrine (NE) turnover rate in rat pineal gland was estimated from the decline of tissue NE levels after the injection of the tyrosine hydroxylase inhibitor alpha-methyl-p-tyrosine. The administration of a single injection of 300 micrograms/Kg of melatonin at the beginning of the scotophase induced, 3 hr later, a significant decrease of pineal NE turnover. The possible direct effect of melatonin on pineal NE release was examined in vitro. Exposure of rat pineal explants previously loaded with 3H-NE to 10(-8)-10(-6) M melatonin decreased significantly 3H-NE release triggered by 60 mM K+. This activity of melatonin was revealed only in pineals excised at night (0000 and 0400, i.e., at the fourth or eighth hours of darkness) and not in those excised in the middle (1400) or late light phase of the daily photoperiod (2000). Melatonin did not modify the spontaneous pineal 3H-NE efflux. Melatonin decreased 3H-NE uptake at a low NE concentration (0.5 microM) in a dose-dependent manner (IC50 identical to 10(-10) M). A kinetic analysis of the pineal NE uptake process indicated that melatonin augmented both Vmax and Km of transmitter uptake. These results suggest that endogenously released melatonin may be a regulatory signal for rat pineal sympathetic synapses.

The effects of alpha-methyl-p-tyrosine pretreatment on ethanol-induced narcosis and hypothermia, as well as in the development of tolerance to these effects in UChA and UChB rats.

We have previously reported that UChA rats (genetically low ethanol consumer) develop tolerance to narcosis time easier than UChB rats (genetically high ethanol consumer). We also have reported that UChA rats develop tolerance to the hypothermic effect of ethanol, while in UChB rats the repeated administration of ethanol induces sensitization towards this effect. In the present paper the effects of alpha-methyl-p-tyrosine (AMPT)--a competitive inhibitor of norepinephrine synthesis--on ethanol-induced narcosis and hypothermia, as well as in the development of tolerance to these effects, were studied in both strains of rats. Results obtained show that AMPT pretreatment induced a significantly higher increase in narcosis time and hypothermia, as well as, greater susceptibility to ethanol toxicity in UChB than UChA rats. Furthermore, the simultaneous treatment with AMPT and ethanol did not change the development of tolerance to narcosis time in both strains and to hypothermia and sensitization in UChA and UChB rats respectively.