Structural basis for activation of the growth hormone-releasing hormone receptor.

Growth hormone-releasing hormone (GHRH) regulates the secretion of growth hormone that virtually controls metabolism and growth of every tissue through its binding to the cognate receptor (GHRHR). Malfunction in GHRHR signaling is associated with abnormal growth, making GHRHR an attractive therapeutic target against dwarfism (e.g., isolated growth hormone deficiency, IGHD), gigantism, lipodystrophy and certain cancers. Here, we report the cryo-electron microscopy (cryo-EM) structure of the human GHRHR bound to its endogenous ligand and the stimulatory G protein at 2.6 Å. This high-resolution structure reveals a characteristic hormone recognition pattern of GHRH by GHRHR, where the α-helical GHRH forms an extensive and continuous network of interactions involving all the extracellular loops (ECLs), all the transmembrane (TM) helices except TM4, and the extracellular domain (ECD) of GHRHR, especially the N-terminus of GHRH that engages a broad set of specific interactions with the receptor. Mutagenesis and molecular dynamics (MD) simulations uncover detailed mechanisms by which IGHD-causing mutations lead to the impairment of GHRHR function. Our findings provide insights into the molecular basis of peptide recognition and receptor activation, thereby facilitating the development of structure-based drug discovery and precision medicine.

Growth hormone-releasing hormone (GHRH) deficiency promotes inflammation-associated carcinogenesis.

The somatotropic axis, in addition to its well-known metabolic and endocrine effects, plays a pivotal role in modulation of inflammation. Moreover, growth hormone (GH)-releasing hormone (GHRH) has been involved in the development of various human tumors. In this work we aimed to investigate the consequences of GHRH deficiency on the development of inflammation-associated colon carcinogenesis in a mouse model of isolated GH deficiency due to generalized ablation of the GHRH gene [GHRH knock out (GHRHKO)]. Homozygous GHRHKO (-/-) male mice and wild type (C57/BL6, +/+) male mice as control group, were used. After azoxymetane (AOM)/dextran sodium sulfate (DSS) treatment -/- mice displayed higher Disease Activity Index (DAI) score, and more marked weight loss compared to +/+ animals. Additionally, -/- mice showed a significant increase in total tumors, in particular of large size predominantly localized in distal colon. In colonic tissue of AOM/DSS-treated -/- mice we found the presence of invasive adenocarcinomas, dysplasia and colitis with mucosal ulceration. Conversely, AOM/DSS-treated +/+ mice showed only presence of adenomas, without invasion of sub-mucosa. Treatment with AOM/DSS significantly increased prostaglandin (PG)E2 and 8-iso-PGF2α levels along with cyclooxygenase-2 (COX-2), tumor necrosis factor (TNF)-α, nuclear factor kappa B (NF-kB) and inducible nitric oxide synthase (iNOS) gene expression, in colon specimens. The degree of increase of all these parameters was more markedly in -/- than +/+ mice. In conclusion, generalized GHRH ablation increases colon carcinogenesis responsiveness in male mice. Whether this results from lack of GH or GHRH remains to be established.

Increased pain and inflammatory sensitivity in growth hormone-releasing hormone (GHRH) knockout mice.

Growth hormone (GH) and GH-releasing hormone (GHRH), in addition to metabolic and endocrine effects, play a role in the modulation of pain and inflammation. We aimed to elucidate the consequences of GHRH deficiency on acute nociceptive stimulation and on both acute and chronic inflammatory stimuli in a mouse model of GH deficiency. Mice with generalized ablation of the GHRH gene (GHRH knock out, GHRHKO, -/-) were compared to wild type (GHRH +/+) mice. Responsiveness to acute nociceptive stimulation and to acute inflammatory stimulation was evaluated by conventional hot plate apparatus and formalin test, respectively. We also evaluated responsiveness to colonic inflammation induced both in vivo, after dextran sodium sulfate (DSS) treatment, or ex vivo, by incubating colon segments with bacterial lipopolysaccaride (LPS). Macroscopical and histological examinations were performed, prostaglandin (PG) E2 and 8-iso-PGF2α levels and cyclooxigenase (COX)-2 and tumor necrosis factor (TNF)-α gene expression were measured. Compared to controls, -/- mice showed decreased response latency during the hot plate test, and increased licking/biting time in formalin test, particularly in the second phase of inflammation. DSS treated -/- mice showed a significant increase of colonic inflammation compared to controls. Moreover DSS treatment increased PGE2 and 8-iso-PGF2α levels, along with COX-2 and TNF-α gene expression more markedly in colon specimens of -/- mice compared to controls. LPS-induced PGE2 and 8-iso-PGF2α production from colonic segments incubated ex vivo was also increased in -/- mice. Generalized GHRH gene ablation increases sensitivity to thermal pain and both acute and persistent inflammatory stimuli in male mice.

Alterations in oxidative, inflammatory and apoptotic events in short-lived and long-lived mice testes.

Aged testes undergo profound histological and morphological alterations leading to a reduced functionality. Here, we investigated whether variations in longevity affect the development of local inflammatory processes, the oxidative state and the occurrence of apoptotic events in the testis. To this aim, well-established mouse models with delayed (growth hormone releasing hormone-knockout and Ames dwarf mice) or accelerated (growth hormone-transgenic mice) aging were used. We hereby show that the testes of short-lived mice show a significant increase in cyclooxygenase 2 expression, PGD2 production, lipid peroxidation, antioxidant enzymes expression, local macrophages and TUNEL-positive germ cells numbers, and the levels of both pro-caspase-3 and cleaved caspase-3. In contrast, although the expression of antioxidant enzymes remained unchanged in testes of long-lived mice, the remainder of the parameters assessed showed a significant reduction. This study provides novel evidence that longevity confers anti-inflammatory, anti-oxidant and anti-apoptotic capacities to the adult testis. Oppositely, short-lived mice suffer testicular inflammatory, oxidative and apoptotic processes.

GH deficiency in a Dalmatian puppy with megaesophagus.

An 8 wk old female Dalmatian weighing .56 kg presented with growth retardation. The puppy exhibited no abnormalities during physical examination other than significantly reduced growth compared with her littermates. Endocrine results suggested pituitary dwarfism. Two wk later, the puppy returned due to the onset of megaesophagus, but the puppy unfortunately died the following morning. This case report describes the diagnosis of dwarfism in a Dalmatian puppy that was caused by growth hormone (GH) deficiency and describes its early clinical manifestations.

Testing for growth hormone deficiency in adults: doing without growth hormone-releasing hormone.

PURPOSE OF REVIEW: This article summarizes recent advances in testing for growth hormone deficiency (GHD) in adults, focusing on critical appraisal of existing growth hormone (GH) provocative tests as well as newer tests in development. RECENT FINDINGS: The diagnosis of GHD can be challenging and often requires the use of GH provocative testing. The most widely validated of these is insulin-induced hypoglycemia (ITT), which requires close supervision and has significant contraindications and side-effects. The arginine-growth hormone-releasing hormone (GHRH) test had become widely used as a safe and accurate alternative to the ITT, but GHRH is currently unavailable for clinical use in the USA. On the basis of review of recent literature we recommend that in the absence of GHRH, glucagon stimulation testing should be the preferred alternative to ITT. Several synthetic GH secretagogues that mimic the gastric peptide ghrelin are currently in development and may become available for use in the diagnosis of GHD in the near future. Other GH provocative tests suitable for use in children lack adequate specificity for the diagnosis of GHD in adults. SUMMARY: Due to the current unavailability of the arginine-GHRH test in the USA, when ITT is contraindicated or impractical we recommend the glucagon stimulation testing as the GH provocative test of choice. There remains a need for a simple, safe and accurate test for the diagnosis of GHD.

Influenza virus-induced glucocorticoid and hypothalamic and lung cytokine mRNA responses in dwarf lit/lit mice.

Influenza virus infection up-regulates cytokines such as interleukin-1beta (IL-1beta) and activates the somatotropic axis and the hypothalamic-pituitary axis. Mice with deficits in growth hormone releasing hormone (GHRH) signaling (lit/lit mice) respond to influenza virus challenge with a progressive decrease in sleep and lower survival rates. Current experiments characterize plasma glucocorticoid responses and hypothalamic and lung mRNA expression of sleep-related genes in lit/lit mice and their heterozygous controls after influenza virus challenge. lit/lit mice had higher basal and post-infection plasma corticosterone levels compared to controls. In contrast, the heterozygous mice increased hypothalamic GHRH-receptor, CRH-type 2 receptor, IL-1beta, and tumor necrosis factor-alpha (TNF-alpha) mRNAs after virus treatment while the lit/lit mice failed to up-regulate these substances. In contrast, lung levels of IL-1beta and TNF-alpha mRNAs were greater in the lit/lit mice. These data are consistent with the hypothesis that the sleep response to influenza infection is mediated, in part, by an up-regulation of hypothalamic sleep-related transcripts and they also show that a primary deficit in GHRH signaling is associated with enhanced corticosterone secretion and attenuated hypothalamic cytokine response to infection.

Hypothalamic growth hormone-releasing hormone (GHRH) deficiency: targeted ablation of GHRH neurons in mice using a viral ion channel transgene.

Animal and clinical models of GHRH excess suggest that GHRH provides an important trophic drive to pituitary somatotrophs. We have adopted a novel approach to silence or ablate GHRH neurons, using a modified H37A variant of the influenza virus M2 protein ((H37A)M2). In mammalian cells, (H37A)M2 forms a high conductance monovalent cation channel that can be blocked by the antiviral drug rimantadine. Transgenic mice with (H37A)M2 expression targeted to GHRH neurons developed postweaning dwarfism with hypothalamic GHRH transcripts detectable by RT-PCR but not by in situ hybridization and immunocytochemistry, suggesting that expression of (H37A)M2 had silenced or ablated virtually all the GHRH cells. GHRH-M2 mice showed marked anterior pituitary hypoplasia with GH deficiency, although GH cells were still present. GHRH-M2 mice were also deficient in prolactin but not TSH. Acute iv injections of GHRH in GHRH-M2 mice elicited a significant GH response, whereas injections of GHRP-6 did not. Twice daily injections of GHRH (100 microg/d) for 7 d in GHRH-M2 mice doubled their pituitary GH but not PRL contents. Rimantadine treatment failed to restore growth or pituitary GH contents. Our results show the importance of GHRH neurons for GH and prolactin production and normal growth.

Ghrelin stimulates GH but not food intake in arcuate nucleus ablated rats.

Ghrelin, an endogenous ligand for the GH secretagogue receptor 1a (GHS-R(1a)), was originally purified from the rat stomach. Ghrelin mRNA and peptide have also been detected in the hypothalamus and pituitary. Ghrelin is a novel acylated peptide that regulates GH release and energy metabolism. GHS-R(1a) mRNA is expressed in the pituitary gland as well as in several areas of the brain including the hypothalamus. In this study, we examined whether ghrelin could stimulate GH secretion and feeding in chronic GHRH, neuropeptide Y, and agouti-related protein deficient rats that were neonatally treated with monosodium glutamate (MSG), which destroys the neurons in the hypothalamic arcuate nucleus (ARC). Intravenous (iv) administration of rat ghrelin (10 micro g/kg body weight) increased plasma GH levels significantly in the normal adult male rats during a GH trough period of pulsatile GH secretion, while iv injection of ghrelin in MSG-treated rats resulted in a markedly attenuated GH response. When rat ghrelin (10 micro g/rat) was administered intracerebroventricular (icv), plasma GH levels were increased comparably in normal control and MSG-treated rats. However, the GH release after icv injection of ghrelin was markedly diminished compared with that after iv administration of a small amount of ghrelin in normal control rats (icv: 10 micro g/rat, iv: approximately 4.0 micro g/rat), indicating that the GH-releasing activity of exogenous ghrelin is route dependent and at least in part via hypothalamic ARC. The icv administration of 1 micro g of ghrelin increased significantly 4-h food intake in normal control, whereas the peptide did not increase food intake in MSG-treated rats, indicating that the feeding response to ghrelin requires intact ARC. Taken together, the primary action of ghrelin on appetite control and GH releasing activity is via the ARC even though it might act on another type of GHS-R besides GHS-R(1a).

Deficiency of growth hormone-releasing hormone signaling is associated with sleep alterations in the dwarf rat.

The somatotropic axis, and particularly growth hormone-releasing hormone (GHRH), is implicated in the regulation of sleep-wake activity. To evaluate sleep in chronic somatotropic deficiency, sleep-wake activity was studied in dwarf (dw/dw) rats that are known to have a defective GHRH signaling mechanism in the pituitary and in normal Lewis rats, the parental strain of the dw/dw rats. In addition, expression of GHRH receptor (GHRH-R) mRNA in the hypothalamus/preoptic region and in the pituitary was also determined by means of reverse transcription-PCR, and GHRH content of the hypothalamus was measured. Hypothalamic/preoptic and pituitary GHRH-R mRNA levels were decreased in the dw/dw rats, indicating deficits in the central GHRHergic transmission. Hypothalamic GHRH content in dw/dw rats was also less than that found in Lewis rats. The dw/dw rats had less spontaneous nonrapid eye movement sleep (NREMS) (light and dark period) and rapid eye movement sleep (REMS) (light period) than did the control Lewis rats. After 4 hr of sleep deprivation, rebound increases in NREMS and REMS were normal in the dw/dw rat. As determined by fast Fourier analysis of the electroencephalogram (EEG), the sleep deprivation-induced enhancements in EEG slow-wave activity in the dw/dw rats were only one-half of the response in the Lewis rats. The results are compared with sleep findings previously obtained in GHRH-deficient transgenic mice. The alterations in NREMS are attributed to the defect in GHRH signaling, whereas the decreases in REMS might result from the growth hormone deficiency in the dw/dw rat.

In vivo semiquantification of hypothalamic growth hormone-releasing hormone (GHRH) output in humans: evidence for relative GHRH deficiency in aging.

GH secretion declines with aging. The neuroendocrine mechanisms of somatopause are uncertain. To semiquantify endogenous hypothalamic GHRH output, we measured the suppressibility of spontaneous and GHRH-stimulated GH secretion by graded doses of a specific competitive GHRH receptor antagonist (N-Ac-Tyr1,D-Arg2)GHRH-(1-29) in healthy young and elderly men. Nocturnal GH was about 30% lower in the elderly than in the young. Graded boluses of GHRH elicited dose-dependent GH responses, with no difference between the two age groups. Graded infusions of GHRH antagonist suppressed GH responses to GHRH in a dose-dependent manner, but with similar potency in both groups. The degree of inhibition depended on the magnitude of GHRH bolus: the dose-inhibition curves for the low GHRH boluses were shifted to the left compared to those with the high GHRH bolus (P = 0.01). Similarly, the dose-inhibition curve for spontaneous GH secretion was shifted to the left for the elderly compared to the young men (P = 0.01). Thus, the model of graded infusions of GHRH antagonist differentiates between different amounts of GHRH presented to the pituitary, permitting semiquantification of the endogenous hypothalamic GHRH output in vivo. Our data suggest that there is an age-dependent decrease in the endogenous hypothalamic GHRH output contributing to the age-associated GH decline.

Elements in the pathophysiology of diminished growth hormone (GH) secretion in aging humans.

Remarkable decreases in growth hormone (GH) secretion accompany healthy aging. The pathophysiology of this hyposomatotropism is confounded by concurrent changes in body composition (with increased visceral fat), physiological declines in estrogen and androgen concentrations, differences in gender responses to aging, and alterations not only in the quantity of GH secreted, but also (as more recently evident) in the orderliness or regularity of the GH release process (e.g., as assessed by approximate entropy). In addition, physical fitness or aerobic capacity also positively modulates GH secretion. Lastly, confounding variables such as altered sleep patterns and nutritional state may contribute to overall regulation of the GH axis in aging. Despite confounding variables, available human experiments suggest partial growth hormone-releasing hormone (GHRH) deficiency in healthy older individuals, presumptively combined with somatostatin excess, and disruption of the moment-to-moment pattern of coordinated and orderly GH release. Here, the authors review these selected facets of recent experimental evaluation of the human GH insulin-like growth factor-I (GH-IGF-I) feedback axis in aging humans.

Functional consequences of the somatopause and its treatment.

The decline in the function of the growth hormone-releasing hormone, growth hormone, insulin-like growth factor (GHRH-GH-IGF) axis has been termed the somatopause. Many of the catabolic sequelae seen in normal aging has been attributed to this decrease in circulating GH and IGF-I. In order to provide hormone replacement therapy for the somatopause, elderly subjects have been treated with GH, IGF-I, or both hormones together. Whereas numerous beneficial effects on body composition, strength, and quality of life have been reported in some studies, other studies have reported only marginal functional improvements. Moreover, it is clear that both hormones can cause significant morbidity.

Non-rapid eye movement sleep is suppressed in transgenic mice with a deficiency in the somatotropic system.

Sleep-wake activity was studied in a transgenic mouse model (TH-hGH) with a deficiency in the somatotropic axis (growth hormone (GH)-releasing hormone (GHRH)-GH-insulin-like growth factor-I (IGF-I)). This dwarf transgenic mouse strain expresses a human GH (hGH) reporter gene linked to 4.8 kb of the rat tyrosine hydroxylase flanking sequence, targeting the production of hGH to sites of tyrosine hydroxylase synthesis in the brain. Endogenous GH and IGF-I are suppressed in these mice, as well as GHRH. Sleep-wake activity (EEG and EMG) was recorded for 2 to 3 days in nine transgenic mice and nine wild-type littermates. Non-rapid eye movement sleep (NREMS) was significantly suppressed during both the light and the dark period in the transgenic mice; rapid eye movement sleep (REMS) was not altered. The results provide evidence that the somatotropic axis contributes to normal sleep regulation.

Physiological regulation of the human growth hormone (GH)-insulin-like growth factor type I (IGF-I) axis: predominant impact of age, obesity, gonadal function, and sleep.

The growth hormone (GH)-insulin-like growth factor type I (IGF-I) axis is subject to exquisite regulation by multiple internal physiological variables and external cues. This review and update summarizes the impact of age, obesity, gonadal function and sleep on the control of GH secretion by the pituitary gland, as regulated by the dominant hypothalamic regulatory peptides, GH-releasing hormone (GHRH) and somatostatin. Available studies show an exponential decline in the calculated daily GH-secretion rate as a function of age in healthy men, such that every 7 years of advancing age beyond age 18-21 results in an approximately 50% decline. There are also strongly negative correlations between the daily GH-secretion rate and indices of obesity, such as the body mass index (BMI). For each increase in BMI of 1.5 kg/m2, there is a 50% decrease in the amount of GH secreted per day. At puberty, and across a span of adult ages, gonadal steroid-hormone concentrations in blood positively determine GH release. In particular, serum estradiol and testosterone concentrations are proportionate to GH-secretory burst mass and mean serum GH concentrations. Deep sleep (stages 3 and 4) is accompanied by markedly increased pulsatile GH secretion that can be accounted for mechanistically by presumptive somatostatin withdrawal combined with hypothalamic GHRH release. Lastly, body composition (especially visceral adiposity) appears to be a dominant negative determinant of GH production, since the relationships between GH secretion and age, testosterone, or sleep are all attenuated or abolished by adiposity. Recent data using pulsatile GHRH treatment or pharmacological methods to reduce somatostatin secretion point to combined defects in GHRH release and somatostatin excess as the most plausible pathophysiology of hyposomatotropism accompanying obesity.

Reproducibility of the growth hormone response to stimulation with growth hormone-releasing hormone plus arginine during lifespan.

The reliability and reproducibility of provocative stimuli of growth hormone (GH) secretion in the diagnosis of GH deficiency are still controversial both in childhood and in adulthood. The combined administration of GH-releasing hormone (GHRH) and arginine (ARG), which likely acts via inhibition of hypothalamic somatostatin release, is one of the most potent stimuli known so far and has been proposed recently as the best test to explore the maximal somatotrope capacity of somatotrope cells. However, it is well known that, usually, provocative stimuli of GH secretion suffer from poor reproducibility and that of the GHRH + ARG test has still to be verified. We aimed to verify the between- and within-subject variability of the GH response to the GHRH + ARG test in normal subjects during their lifespan as well as in hypopituitaric patients with GH deficiency (GHD). In 10 normal children (C: six male and four female, age 12.3 +/- 0.9 years, body mass index (BMI) = 16.6 +/- 0.7 kg/m2, pubertal stages I-III), 18 normal young adults (Y: ten male and eight female, age 31.1 +/- 1.3 years, BMI = 21.4 +/- 0.4 kg/m2), 12 normal elderly subjects (E: two male and ten female, age 74.4 +/- 1.8 years, BMI= 22.6 +/- 0.6 kg/m2) and 15 panhypopituitaric GH-deficient patients (GHD: nine male and six female, age 40.9 +/- 4.1 years, BMI= 22.7 +/- 1.0 kg/m2), we studied the inter- and intra-individual variability of the GH response to GHRH (1 microg/kg i.v.) + ARG (0.5 g/kg i.v.) in two different sessions at least 3 days apart. The GH responses to GHRH + ARG in C (1st vs 2nd session: 61.6 +/- 8.1 vs 66.5 +/- 9.4 microg/l), Y (70.4 +/- 10.1 vs 76.2 10.7 microg/l) and E (57.9 14.8 vs 52.1 +/- 8.0 microg/l) were similar and reproducible in all groups. The somatotrope responsiveness to GHRH + ARG also showed a limited within-subject variability (r = 0.71, 0.90 and 0.89 and p < 0.02, 0.0005 and 0.0005 for C, Y and E, respectively). Similarly in GHD, the GH response to the GHRH + ARG test showed a good inter- (1st vs 2nd session: 2.3 +/- 0.5 vs 2.2 +/- 0.6 microg/l) and intra-individual reproducibility (r = 0.70, p < 0.005). The GHRH + ARG-induced GH responses in GHD were markedly lower (p < 0.0005) than those in age-matched controls and no overlap was found between GH peak responses in GHD and normal subjects. In normal subjects, the GH response to GHRH + ARG is very marked, independent of age and shows limited inter- and intra-individual variability. The GH response to the GHRH + ARG test is strikingly reduced in panhypopituitaric patients with GHD, in whom the low somatotrope responsiveness is reproducible. Thus, these findings strengthen the hypothesis that GHRH + ARG should be considered the most reliable test to evaluate the maximal secretory capacity of somatotrope cells and to distinguish normal subjects from GHD patients in adulthood.

Growth hormone-releasing hormone depletion in the female rat: similarities to aging.

The age-related decline in growth hormone (GH) secretion has been largely attributed to age-related degeneration of hypothalamic growth hormone-releasing hormone (GHRH)-producing neurons. GH decline has recently been linked to age-related bone changes in humans. Bone loss and decreased bone strength are common in aging rats and humans, but density of remaining mineral tissue is known to be increased. The effect of induced hypothalamic GHRH deficiency on bone was assessed, and similarities between bone changes encountered and those taking place in aging were identified. Female rats received monosodium glutamate (MSG) following birth, and they were euthanized at 19 weeks of age. Femurs from MSG-treated rats had greater mineral density (p < .05), greater mineral/matrix ratio (p < .01), lower mineral apposition rate (p < .005), and lower bone formation rate (p < .05). These results suggest that hypothalamic GHRH decline plays a substantial role in the development of bone pathology similar to that observed in aging individuals.

Somatotropic dysfunction in growth hormone-releasing hormone-deprived neonatal rats: effect of growth hormone replacement therapy.

In a previous work, we reported that passive immunization with anti-growth hormone-releasing hormone (GHRH) antibodies (GHRH-Ab) in neonatal rats caused disruption of somatotropic function that was still present 60 d posttreatment. We studied the reversibility of this condition by growth hormone (GH) replacement therapy. Neonatal rats received GHRH-Ab (50 microL/rat, s.c.) or normal rabbit serum every second day from birth up to postnatal d 10 and received hGH (0.4 microgram/g body weight, s.c., b.i.d.) or vehicle in a 2 x 2 factorial design. Animals were studied on d 11 of age. In GHRH-Ab-treated rats, GH therapy 1) counteracted the reduced body weight and low plasma IGF-I levels; 2) failed to modify the reduced pituitary weight and GH content; 3) further reduced the low plasma GH levels; 4) partially restored the defective GH responsiveness to GHRH; 5) failed to modify the reduced hypothalamic somatostatin and increased GHRH gene expression in the hypothalamus; and 6) reverted the decreased pituitary somatostatin binding. Morphologic and morphometric evaluation of the pituitary gland from GHRH-AB+GH pups showed that the number of GH-labeled structures was lower than in normal rat serum-GH-treated pups, whereas the total GH immunoreactivity per unit surface, an index of intracellular hormone concentration, was slightly higher than in vehicle-GH or GHRH-Ab pups. As determined by electron microscopy, somatotropes from GHRH-Ab+GH pups had morphologic features of high cellular activity. It appears that in GHRH-deprived pups GH replacement therapy can normalize most but not all altered indices of the somatotropic function.(ABSTRACT TRUNCATED AT 250 WORDS)