Feasibility of whole genome and transcriptome profiling in pediatric and young adult cancers.

The utility of cancer whole genome and transcriptome sequencing (cWGTS) in oncology is increasingly recognized. However, implementation of cWGTS is challenged by the need to deliver results within clinically relevant timeframes, concerns about assay sensitivity, reporting and prioritization of findings. In a prospective research study we develop a workflow that reports comprehensive cWGTS results in 9 days. Comparison of cWGTS to diagnostic panel assays demonstrates the potential of cWGTS to capture all clinically reported mutations with comparable sensitivity in a single workflow. Benchmarking identifies a minimum of 80× as optimal depth for clinical WGS sequencing. Integration of germline, somatic DNA and RNA-seq data enable data-driven variant prioritization and reporting, with oncogenic findings reported in 54% more patients than standard of care. These results establish key technical considerations for the implementation of cWGTS as an integrated test in clinical oncology.

Glycine modulation of the phencyclidine binding site in mammalian brain.

Neurophysiological studies have shown that glycine potentiates the NMDA response in cultured neurons by a strychnine-insensitive mechanism. Autoradiographic data have demonstrated a correspondence between strychnine-insensitive [3H]glycine binding sites and NMDA-sensitive [3H]glutamate binding sites. Here we report that in synaptic plasma membranes from rat brain, the binding of a PCP analog, [3H]TCP, was enhanced more than 5-fold by 1 microM glycine. This glycine stimulation of binding of [3H]TCP was blocked by the competitive NMDA-receptor antagonist, D-AP7. These data provide support for the hypothesis that a unique amino acid recognition site is associated with the proposed NMDA/PCP receptor complex in brain.

Neuropeptide Y in the rat nucleus accumbens: ultrastructural localization in aspiny neurons receiving synaptic input from GABAergic terminals.

The ultrastructure, afferent input, and sites of termination of neurons containing neuropeptide Y-like immunoreactivity (NPY-LI) were examined in the adult rat nucleus accumbens by using the peroxidase-antiperoxidase (PAP) method. The NPY-LI was seen in sparsely distributed, spindle-shaped perikarya having cross-sectional diameters of 15-20 microns. These perikarya exhibited highly invaginated nuclear membranes and thin rims of cytoplasm containing Golgi lamellae, dense-core vesicles, and other organelles. A few large, principally aspiny, dendrites also showed NPY-LI. The dendrites received synaptic input from unlabeled terminals forming both symmetric and asymmetric junctions. Immunolabeling for NPY was evident in other processes that were not clearly differentiated as dendrites or axons. These were seen primarily near glial processes and the basal laminae of blood vessels. A few myelinated and many unmyelinated axons and axon terminals also were labeled for NPY. These terminals contained numerous, small (40-60 nm), clear and one or more large (80-100 nm) dense core vesicles. Forty-seven percent (27 out of 57) of the terminals containing NPY-LI formed symmetric junctions with unlabeled dendrites or dendritic spines. The remainder lacked recognizable densities within single planes of section. The neurons exhibiting NPY-LI in the nucleus accumbens were characterized further with respect to their afferent input from terminals labeled for the GABA-synthesizing enzyme, glutamic acid decarboxylase (GAD). Immunogold labeling of a rabbit antiserum against NPY and PAP labeling for a sheep antiserum to GAD were sequentially applied to the same sections. The GAD-labeled terminals formed symmetric junctions primarily with the more numerous unlabeled dendrites. However, a few synaptic junctions also were detected between the GAD-labeled terminals and dendrites showing immunogold labeling for NPY. We conclude (1) that in the rat nucleus accumbens, NPY-LI is found principally in neurons of the aspiny type and (2) that the output from these presumably intrinsic neurons to other neighboring neurons or blood vessels is at least partially modulated by GABA.

Localization of receptors for bombesin-like peptides in the rat brain.

BN-like peptides and receptors are present in discrete areas of the mammalian brain. By radioimmunoassay, endogenous BN/GRP, neuromedin B, and ranatensin-like peptides are present in the rat brain. High-to-moderate concentrations of BN/GRP are present in the rat hypothalamus and thalamus, whereas moderate-to-high densities of neuromedin B and ranatensin-like peptides are present in the olfactory bulb and hippocampus, as well as in the hypothalamus and thalamus. While the distribution of neuromedin B and ranatensin-like peptides appears similar, it is distinct from that of BN/GRP. When released from CNS neurons, these peptides may interact with receptors for BN-like peptides. BN, GRP, ranatensin, and neuromedin B inhibit specific [125I-Tyr4]BN binding with high affinity. By use of in vitro autoradiographic techniques to detect binding of [125I-Tyr4]BN to receptors for BN-like peptides, high grain densities were found in the olfactory bulb and tubercle, the nucleus accumbens, the suprachiasmatic and paraventricular nucleus of the hypothalamus, the central medial and paraventricular thalamic nuclei, the hippocampus, the dentate gyrus, and the amygdala of the rat brain. Some of these receptors may be biologically active and mediate the biological effects of BN-like peptides. For example, when BN is directly injected into the nucleus accumbens, pronounced grooming results and the effects caused by BN are reversed by spantide and [D-Phe12]BN. Thus, the putative BN receptor antagonists may serve as useful agents to investigate the biological significance of BN-like peptides in the CNS.

Alpha-melanocyte-stimulating hormone structure-activity studies: comparative analysis of melanotropic and CNS bioactivities.

The structure-activity relationships in vitro of alpha-MSH (alpha-melanocyte-stimulating hormone, alpha-melanotropin) analogs as determined on normal and transformed (melanoma cell) melanocyte bioassays are summarized. Specifically, the characterization of potent and metabolically stable melanotropic agonist analogs and a newly discovered antagonist of alpha-MSH are highlighted. Comparison of these data versus the known structure-activity relationships of alpha-MSH related to CNS bioactivities suggests the existence of nonclassical alpha-MSH receptor-mediated pathways or, perhaps, a yet undefined endogenous neuropeptidergic pathway(s) having different selectivities for alpha-MSH analogs. In summary, several of the alpha-MSH analogs reported here may be useful molecular probes in future strategies aimed at the identification and systematic characterization of both peripheral and central alpha-MSH receptors.

Biochemical and behavioral effects of sigma and PCP ligands.

The purpose of this study was to examine the binding and behavioral effects mediated by PCP and sigma receptors in the rat. From the radioreceptor assays, it was possible to characterize two binding sites that interact with PCP and sigma ligands. The two sites, a PCP and sigma receptor, could be differentiated based on drug selectivity and potency. In the behavioral assays, MK-801, which bound preferentially to the PCP receptor, and 1,3-di-0-tolylguanidine, which bound preferentially to the sigma receptor, induced sniffing, rearing, circling, backpedaling, and weaving behavior. These results indicate that there are distinct PCP and sigma receptors that are both involved in mediating stereotyped behavior and ataxia in the rat.

Dopaminergic agents selectively alter the post-translational processing of beta-endorphin in the intermediate pituitary of the rat.

The present study examines whether sustained changes in the biosynthetic activity of the intermediate pituitary, induced pharmacologically by treating rats with dopamine receptor ligands, alters the post-translational processing of beta-endorphin (beta-END). Separation of the individual molecular forms of beta-END using ion exchange chromatography revealed that beta-END is processed to alpha-N-acetylated and nonacetylated forms of beta-END-(1-31), beta-END-(1-27) and beta-END-(1-26) in the neurointermediate lobe (NIL). Chronic treatment with D-2, but not D-1, dopamine receptor antagonists elevated total beta-END immunoreactivity (i beta-END) in the NIL but this increase was predominantly attributable to elevations in the concentrations of N-acetyl-beta-END-(1-31) and N-acetyl-beta-END-(1-27). In contrast, N-acetyl-beta-END-(1-26) was not significantly affected. The dopaminergic agonist, bromocriptine, had the opposite effect; it lowered total i beta-END levels, depleting N-acetyl-beta-END-(1-31) and N-acetyl-beta-END-(1-27) to a greater extent than N-acetyl-beta-END-(1-26). beta-END peptides were released in vitro in the same relative proportion as they were contained in the NIL suggesting that individual molecular forms of beta-END are not released preferentially. i gamma-END levels also were modulated by dopaminergic agents but the processing of gamma-END and alpha-END was not altered. Acute haloperidol treatment depleted i gamma-END levels but did not affect the pattern of beta-END peptides in the NIL. These results indicate that dopaminergic agents influence not only the secretion but also the post-translational processing of pituitary beta-END.

CRF and cAMP regulation of POMC gene expression in corticotrophic tumor cells.

The effects of corticotropin releasing factor (CRF) on pro-opiomelanocortin (POMC) gene expression were investigated in an anterior pituitary corticotrophic tumor cell line, AtT-20/D16-16. The results of mRNA dot blot hybridization assays suggested that CRF, at a concentration of 10(-7) M, positively regulates the expression of the POMC gene in AtT-20 cells in a concentration-dependent fashion. Evaluation of the time course of this effect indicated that CRF had a biphasic mode of action. CRF and alpha-amanitin (inhibitor of RNA polymerase II activity) were also found to affect POMC mRNA levels in a concentration-dependent fashion. Eight-bromo-cyclic adenosine monophosphate (8-Br-cAMP) produced biphasic effects on POMC mRNA levels, supporting evidence of a role for cAMP as a second messenger in the regulation of POMC gene expression. It was also found that alpha-amanitin negatively regulated basal and CRF-stimulated POMC mRNA levels at both the 2 hr and 24 hr time periods, supporting evidence for positive regulation of POMC by CRF at the transcriptional level.

Selective memory impairment by phencyclidine in rats.

Phencyclidine (PCP) users sometimes report lack of recall of events occurring while they are under the influence of the drug. The present experiment was designed to test whether rats remember information learned after PCP administration. Rats were trained to choose one arm of a T-maze to obtain a food reward. The following day they were injected with either PCP (1 mg/kg) or vehicle and trained to choose the opposite arm for a reward (reversal learning). A third group of rats received neither injections nor training on the second day. On the third day, all rats were tested for their preference of maze arms. Rats who had been injected with saline before reversal learning chose the arm rewarded during the reversal, while rats receiving PCP on the second day chose randomly. The rats which did not learn the reversal chose the arm learned on day 1. These results indicate that while PCP did not interfere with the rats ability to learn, it interfered with long-term storage of information.

Activation of distinct second messenger systems in anterior pituitary corticotrophic tumor cells alters the phosphorylation states of both shared and distinct cytosolic proteins.

The purpose of the present study was to investigate the effects of activation of different second messenger systems on protein phosphorylation in pituitary corticotrophic tumor cells (AtT-20/D16-16). Using two-dimensional gel analysis of cytosolic extracts from AtT-20 cells, several phosphoproteins exhibited alterations in 32P incorporation in response to stimulation of the cells with either forskolin--an activator of adenylate cyclase--or 12-O-tetradecanoyl phorbol-13-acetate (TPA)--a tumor promoting phorbol ester linked to protein kinase C activation. Alterations in phosphorylation levels were seen for phosphoproteins of the following apparent molecular weights and pIs: 87 kDa (pI 4.4-4.6), 67 kDa (pI 4.7-4.9), 43 kDa (pI 4.8-5.0), 39 kDa (pI 4.9-5.1), 33 kDa (pI 4.8-5.0), 19.5 kDa (pI 5.7-5.9), 19 kDa (pI 5.8-6.0), 16 kDa (pI 5.2-5.4) and 14 kDa (pI 5.1-5.3). For individual phosphoproteins, 32P incorporation varied over time and was also modulated by concentrations of Ca2+ and Mg2+ in the incubation medium. Treatment of the cells with forskolin led to statistically significant changes in the phosphorylation states of the 19.5 and 14 kDa proteins. Treatment of the cells with TPA also produced statistically significant changes in the 19.5 and 14 kDa proteins but, in addition, the 87 kDa, the 39 kDa and the 16 kDa phosphoproteins also exhibited significant changes. Alterations in the phosphorylation states of the 19.5 and the 14 kDa proteins were significantly correlated with alterations in beta-endorphin release from the cells. The primary finding of the present study was that activation of distinct second messenger systems can lead to alterations in the phosphorylation states of both shared and distinct phosphoproteins.

Neuropeptide Y levels in microdissected regions of the hypothalamus and in vitro release in response to KCl and prostaglandin E2: effects of castration.

Intracerebroventricular administration of neuropeptide Y (NPY) has been shown to modify LH secretion, with the direction of the response dependent on the steroid background. To study further the role of gonadal steroids in the regulation of NPY secretion, the basal and KCl-evoked release of NPY from the medial basal hypothalamus (MBH) of intact and castrated male rats was assessed twice with the use of an in vitro incubation system. In each experiment, the amounts of NPY released in response to a 15-min pulse of KCl (45 mM) were significantly smaller from the MBH of castrated rats than of intact rats (P less than 0.05). Next, to assess the possible effects of prostaglandin E2 (PGE2), the MBH were exposed in a similar manner to two 15-min pulses, 30 min apart, of 0.568 and 56.8 mumol PGE2. Unlike KCl, PGE2 failed to stimulate NPY release from the MBH of either intact or castrated rats. However, a similar 56.8 mumol concentration of PGE2 was effective in stimulating the release of LHRH. We next examined the effects of castration on NPY levels in several microdissected regions of the hypothalamus. Whereas NPY concentrations were unchanged in the medial preoptic area, paraventricular nucleus and dorsomedial nucleus, NPY levels were significantly decreased in the median eminence, arcuate nucleus, and ventromedial nucleus 2 weeks after castration. These studies show that KCl can stimulate NPY release from the MBH in vitro, like that of LHRH, the KCl-induced NPY response is significantly smaller from the MBH of castrated than intact males, castration can significantly reduce the levels of NPY in the median eminence, arcuate nucleus, and ventromedial nucleus, thereby suggesting that testicular secretions may modulate NPY levels and release from the MBH, and because PGE2 stimulated the release of LHRH but not of NPY, separate regulatory neural events may underlie the secretion of these two neuropeptides.

Histological evaluation of the dopaminergic regulation of proopiomelanocortin gene expression in the intermediate lobe of the rat pituitary, involving in situ hybridization and [3H]thymidine uptake measurement.

The melanotroph, a polyhedral secretory cell with an ovoid smooth nucleus, is the primary cell type of the intermediate lobe (IL) of the rat pituitary. The melanotrophs are not uniform, but differ in the tinctorial properties of their cytoplasm; some cells appear distinctly darker, others lighter, and cells staining in intermediate shades are also found. In addition, in situ hybridization using proopiomelanocortin (POMC) probes shows an uneven distribution of POMC mRNA among melanotrophs, indicating that different cells maintain different levels of biosynthetic activity. Dopaminergic drugs known to alter the secretion of POMC-related peptides from the IL produced parallel changes in histological staining properties and the amount of POMC mRNA per cell, as determined by in situ hybridization. Acute bromocriptine treatment (6 h) produced a dramatic reduction in grain counts over melanotroph cytoplasm (to 10% of the control levels). A similar reduction persisted after chronic treatment. Six hours after a single haloperidol injection, the grain counts were 180% of control levels. After chronic haloperidol treatment, they were further elevated to 300% of control levels. Chronic bromocriptine and haloperidol treatment also changed the thickness of the IL. Bromocriptine reduced and haloperidol treatment increased the number of cell layers in the IL by changing the rate of cell proliferation. Thus, haloperidol treatment significantly increased and bromocriptine treatment significantly decreased the number of melanotrophs labeled by [3H]thymidine. The mitotic index followed the same trend. These results suggest that dopamine regulation of the IL acts by two different mechanisms: POMC gene expression and cellular proliferation. The change in POMC gene expression is the cell's first rapid response. The influence on the cell cycle appears after subchronic and chronic treatment.

Autoradiographic distribution of non-dopaminergic binding sites labeled by [3H]haloperidol in rat brain.

The regional distribution of binding sites labeled by [3H]haloperidol, in the presence of excess spiroperidol, was compared to the regional distribution of receptors labeled by [3H]SCH 23390 and [3H]sulpiride, [3H]SCH 23390 and [3H]sulpiride labeled distinct nuclei, such as the olfactory tubercle, caudate, globus pallidus, substantia nigra, and inferior and superior colliculi. In contrast, the distribution of binding sites labeled by [3H]haloperidol, in the presence of excess spiroperidol, were much more extensive. Some areas containing the highest density of sites labeled by [3H]haloperidol were the external plexiform layer of the olfactory bulb, the cerebral cortex, the paraventricular nuclei, the interpeduncular nucleus and the superior colliculus. The distribution of non-dopaminergic binding sites labeled by haloperidol was clearly quite different from that labeled by dopaminergic ligands.

A developmental influence of substance P on its receptors.

Events which alter the chemical environment of the developing nervous system have long-term consequences for neural function and behavior. Neonatal exposure to a peptide neurotransmitter, substance P (SP), increased the number of SP binding sites in the adult rat salivary glands, without altering their affinity. The neonatal treatment also increased the binding capacity for SP of several brain regions, but had no effect on SP levels in the brain. The increase in SP binding sites may be responsible for increases in sensitivity to SP in adults after neonatal treatment with the peptide.

An endogenous ligand for the sigma opioid binding site.

It had been suggested that phencyclidine (PCP) and sigma opioids exert their similar psychotomimetic effects through a common receptor. Recently, however, there have been several reports demonstrating significant differences between the binding of PCP and SKF 10,047, a sigma opioid agonist, which suggests that there may be distinct PCP and sigma opioid receptors. If these differences in binding represent different receptors, then there may be different endogenous ligands for each receptor. Using porcine brains, which have already been used to isolate and purify an endogenous ligand for the PCP receptor, another factor has been isolated that inhibited the binding of [3H]-(+)SKF 10,047 and not the binding of [3H]-PCP. This factor appears to be a peptide or protein because incubation of the active fraction with pronase, a nonspecific peptidase, eliminated the ability of the porcine fractions to inhibit the binding of [3H]-(+)SKF 10,047. These findings suggest the existence of an endogenous ligand for sigma opioid receptors, which is different from the previously identified endogenous ligand for PCP receptors.

PCP-induced alterations in cerebral glucose utilization in rat brain: blockade by metaphit, a PCP-receptor-acylating agent.

The effects of phencyclidine (PCP) on regional cerebral glucose utilization was determined by using quantitative autoradiography with [14C]-2-deoxyglucose. PCP increased brain metabolism in selected areas of cortex, particularly limbic, and in the basal ganglia and thalamus, whereas the drug decreased metabolism in areas related to audition. These results are consistent with the known physiology of central PCP neurons and may help to suggest brain areas involved in PCP-mediated actions. Moreover, based on the behavioral similarities between PCP psychosis and an acute schizophrenic episode, these data may be relevant to the understanding of schizophrenia. The PCP-receptor-acylating agent, metaphit, blocked most of these PCP actions. In addition, metaphit by itself was found to diminish glucose utilization rather uniformly throughout brain. These results indicate an antagonist effect of metaphit on the PCP system and suggest a widespread action of metaphit, putatively at a PCP-related site, possibly in connection with the N-methyl-D-aspartate (NMDA) receptor.

Phencyclidine. Physiological actions, interactions with excitatory amino acids and endogenous ligands.

Phenycyclidine (PCP) produces many profound effects in the central nervous system. PCP has numerous behavioral and neurochemical effects such as inhibiting the uptake and facilitating the release of dopamine, serotonin, and norepinephrine. PCP also interacts with sigma, mu opioid, muscarinic, and nicotinic receptors. However, the psychotomimetic effects induced by PCP are believed to be mediated by specific PCP receptors, where PCP binds with greater potency than sigma compounds. Electrophysiological, behavioral, and neuro-chemical evidence strongly suggests that at least some of the many PCP actions result from antagonism of excitatory amino acid-induced responses via PCP receptors. The recent isolation and partial characterization of the alpha and beta endopsychosins and the identification of other endogenous ligands for the PCP and sigma receptors, is another promising area of research in the elucidation of the physiological role of an endogenous PCP and sigma system.

Thymosin fraction 5 stimulates secretion of immunoreactive beta-endorphin in mouse corticotropic tumor cells.

In addition to reconstituting immune competence, the thymus gland preparation, thymosin fraction 5 (TSN-5), has recently been shown to stimulate secretion of hormones from the hypothalamic-pituitary adrenal axis in vivo and from pituitary corticotropes in vitro. The purpose of the present study was to investigate the effects of TSN-5 on secretion of immunoreactive beta-endorphin (i beta-E) by mouse corticotropic tumor cells. The release of i beta-E by AtT-20 pituitary tumor cells was increased in a dose-dependent manner by concentrations of 30-600 micrograms/ml of TSN-5, whereas concentrations greater than 1,000 micrograms/ml were increasingly less effective in stimulating secretion. TSN-5 (600 micrograms/ml) significantly stimulated i beta-E release within 7 min; maximal secretory responses (up to 275% of control release) occurred by 4 hr. The secretory response of AtT-20 cells to 600 micrograms/ml TSN-5 (37.9 +/- 2.0 vs. 16.1 +/- 1.0 ng i beta-E/ml/4 hr, mean +/- SE) was similar in magnitude to release evoked by 0.1 microM corticotropin-releasing factor (CRF). Combining TSN-5 and CRF treatments increased secretion of i beta-E to nearly 600% of control levels, an effect greater than an additive influence of the two independent treatments. Whereas CRF treatment reduced the levels of i beta-E in AtT-20 cell extracts after 24-hr treatment by 45% (231.8 +/- 24.7 vs. 417.2 +/- 17.8 ng i beta-E/mg protein, CRF vs. vehicle treatments, respectively), TSN-5 did not significantly alter cellular hormone content. Neither TSN-alpha 1 nor TSN-beta 4, two of the component peptides of TSN-5, affected basal or CRF-stimulated release of i beta-E, indicating that an unidentified constituent(s) is corticotropic.(ABSTRACT TRUNCATED AT 250 WORDS)