Analysis of germline CDKN1C (p57KIP2) mutations in familial and sporadic Beckwith-Wiedemann syndrome (BWS) provides a novel genotype-phenotype correlation.

Beckwith-Wiedemann syndrome (BWS) is a human imprinting disorder with a variable phenotype. The major features are anterior abdominal wall defects including exomphalos (omphalocele), pre- and postnatal overgrowth, and macroglossia. Additional less frequent complications include specific developmental defects and a predisposition to embryonal tumours. BWS is genetically heterogeneous and epigenetic changes in the IGF2/H19 genes resulting in overexpression of IGF2 have been implicated in many cases. Recently germline mutations in the cyclin dependent kinase inhibitor gene CDKN1C (p57KIP2) have been reported in a variable minority of BWS patients. We have investigated a large series of familial and sporadic BWS patients for evidence of CDKN1C mutations by direct gene sequencing. A total of 70 patients with classical BWS were investigated; 54 were sporadic with no evidence of UPD and 16 were familial from seven kindreds. Novel germline CDKN1C mutations were identified in five probands, 3/7 (43%) familial cases and 2/54 (4%) sporadic cases. There was no association between germline CDKN1C mutations and IGF2 or H19 epigenotype abnormalities. The clinical phenotype of 13 BWS patients with germline CDKN1C mutations was compared to that of BWS patients with other defined types of molecular pathology. This showed a significantly higher frequency of exomphalos in the CDKN1C mutation cases (11/13) than in patients with an imprinting centre defect (associated with biallelic IGF2 expression and H19 silencing) (0/5, p<0.005) or patients with uniparental disomy (0/9, p<0.005). However, there was no association between germline CDKN1C mutations and risk of embryonal tumours. No CDKN1C mutations were identified in six non-BWS patients with overgrowth and Wilms tumour. These findings (1) show that germline CDKN1C mutations are a frequent cause of familial but not sporadic BWS, (2) suggest that CDKN1C mutations probably cause BWS independently of changes in IGF2/H19 imprinting, (3) provide evidence that aspects of the BWS phenotype may be correlated with the involvement of specific imprinted genes, and (4) link genotype-phenotype relationships in BWS and the results of murine experimental models of BWS.     

A novel TRH analog, Glp-Asn-Pro-D-Tyr-D-TrpNH2, binds to [3H][3-Me-His2]TRH-labelled sites in rat hippocampus and cortex but not pituitary or heterologous cells expressing TRHR1 or TRHR2

Glp-Asn-Pro-D-Tyr-D-TrpNH(2) is a novel synthetic peptide that mimics and amplifies central actions of thyrotropin-releasing hormone (TRH) in rat without releasing TSH. The aim of this study was to compare the binding properties of this pentapeptide and its all-L counterpart (Glp-Asn-Pro-Tyr-TrpNH(2)) to TRH receptors in native rat brain tissue and cells expressing the two TRH receptor subtypes identified in rat to date, namely TRHR1 and TRHR2. Radioligand binding studies were carried out using [(3)H][3-Me-His(2)]TRH to label receptors in hippocampal, cortical and pituitary tissue, GH4 pituitary cells, as well as CHO cells expressing TRHR1 and/or TRHR2. In situ hybridization studies suggest that cortex expresses primarily TRHR2 mRNA, hippocampus primarily TRHR1 mRNA and pituitary exclusively TRHR1 mRNA. Competition experiments showed [3-Me-His(2)]TRH potently displaced [(3)H][3-Me-His(2)]TRH binding from all tissues/cells investigated. Glp-Asn-Pro-D-Tyr-D-TrpNH(2) in concentrations up to 10(-5)M did not displace [(3)H][3-Me-His(2)]TRH binding to membranes derived from GH4 cells or CHO-TRHR1 cells, consistent with its lack of binding to pituitary membranes and TSH-releasing activity. Similar results were obtained for the corresponding all-L peptide. In contrast, both pentapeptides displaced binding from rat hippocampal membranes (pIC(50) Glp-Asn-Pro-D-Tyr-D-TrpNH(2): 7.7+/-0.2; pIC(50) Glp-Asn-Pro-Tyr-TrpNH(2): 6.6+/-0.2), analogous to cortical membranes (pIC(50) Glp-Asn-Pro-D-Tyr-D-TrpNH(2): 7.8+/-0.2; pIC(50) Glp-Asn-Pro-Tyr-TrpNH(2): 6.6+/-0.2). Neither peptide, however, displaced [(3)H][3-Me-His(2)]TRH binding to CHO-TRHR2. Thus, this study reveals for the first time significant differences in the binding properties of native and heterologously expressed TRH receptors. Also, the results raise the possibility that Glp-Asn-Pro-D-Tyr-D-TrpNH(2) is not displacing [(3)H][3-Me-His(2)]TRH from a known TRH receptor in rat cortex, but rather a hitherto unidentified TRH receptor.     

Application of the 2-deoxy-(1-14C) glucose method to the study of suprachiasmatic nucleus activity and functions: phasic luteinizing hormone secretion and serotonin innervation

Summary A semi-quantitative analysis of 2-deoxy-(1-¹⁴C) glucose (2 DG) incorporation in the supra-chiasmatic nucleus (SCN) was performed in male, castrated female, and castrated oestradiol treated female rats (OVX-E2) during the light period (from 9.00 h to 18.00 h). Unilateral orbital enucleation was carried out on each group. In males and castrated females the 2 DG incorporation in the rostral and caudal SCN was constant throughout the studied period. On the contrary, in OVX-E2 rats, which display a clear cut circadian rhythm of LH secretion, the 2 DG incorporation in the rostral SCN was higher in the afternoon than in the morning. After serotonin (5 HT) depletion, the level of morning labelling reached that of the afternoon. These results suggest the existence of a steroid-linked rhythm of SCN metabolic activity during the light period which might be related to the LH surge and dependent on 5 HT innervation.Preliminary data from this investigation were presented at The Serotoninergic Neuron Meeting, Marseille, July 1980     

Calcium entry via ORAI1 regulates glioblastoma cell proliferation and apoptosis

Introduction

Calcium entry plays a critical role in the proliferation and survival of certain tumors. Ca²⁺ release activated Ca²⁺ (CRAC) channels constitute one of the most important pathways for calcium entry especially that of store-operated calcium entry (SOCE). ORAI1 and stromal interaction molecule1 (STIM1) are essential protein components of CRAC channels. In this study we tested the effect of inhibiting CRAC through ORAI1 and STIM1 on glioblastoma multiforme (GBM) tumor cell proliferation and survival.

Methods

Two glioblastoma cell lines, C6 (rat) and U251 (human), were used in the study. ORAI1 and STIM1 expressions were examined using Western blot and immunohistochemistry. CRAC channel activity and its components were inhibited with ion channel blockers and using siRNA knockdown. Changes in intracellular calcium concentration were recorded using Fura-2 fluorescent calcium imaging. Cell proliferation and apoptosis were examined using MTS and TUNEL assays, respectively.

Results

CRAC blockers, such as SKF-96365 (1-[2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl) propoxy]ethyl-1H-imidazole), 2-aminoethoxydiphenyl borate (2-APB) and Diethylstilbestrol (DES), inhibited cell proliferations and SOCE in GBM cells. Knockdown of ORAI1 and STIM1 proteins using siRNA significantly inhibited C6 cell proliferation and SOCE compared with those in control cells, and a more significant effect was observed in cells with ORAI1 siRNA knockdown than that of STIM1-treated cells. Both CRAC blockers and siRNA treatments increased apoptosis in C-6 cells compared with control.

Conclusion

Calcium entry via ORAI1 and CRAC channels are important for GBM proliferation and survival.     

Study of the activation mechanism of human GRF(1-29)NH2 on rat mast cell histamine release

Human growth releasing factor (GRF) (1–29)NH₂ releases histamine from pleural and peritoneal rat mast cells by a non cytotoxic and non immunological mechanism. Pretreatment of cells with pertussis toxin markedly inhibits the secretion, suggesting a possible function of a Gi-protein in the activation pathway. In order to determine the role of cAMP on GRF mediated secretion, mast cells were preincubated with isobutylmethylxanthine (IBMX) or cholera toxin, since both drugs greatly and enhance cAMP levels. IBMX inhibits mediator secretion while, in contrast, cholera toxin is ineffective to modify histamine release. The PKC activator TPA amplifies the response of mast cells to human GRF, shifting the dose-response curve to the left. The pretreatment of mast cells with the phosphatase inhibitor okadaic acid exerts no effect on the doseresponse function curve to GRF. The response to human GRF does not depend on extracellular calcium, but there is a good correlation between the percent of histamine released and⁴⁵calcium uptake. The kinetic of calcium uptake is fast, maximum uptake being reached in 30 seconds.Abbreviations GRF (growth releasing factor) - PTX (pertussis toxin) - CTX (cholera toxin) - OA (okadaic acid) - TPA (12-Otetradecanoyl-phorbol-13-acetate) - IBMX (isobutyl-methylxanthine)     

A comparative evaluation of microarray slides as substrates for the development of protease assay biosensors

The application of commercially available microarray slides as substrates for fluorogenic protease assays has been explored in terms of binding efficiency, stability, and activity. A fluorescent, biotinylated substrate for botulinum neurotoxin A (BoNTA) was attached via self-assembled monolayer of Streptavidin to amine-reactive aldehyde, epoxy, hydrogel, and polymer slides. Nexterion Slide P® was found to have optimal protein binding efficiency and stability of the slides examined. Addition of glycerol to the printing buffer improved spot morphology significantly and polyvinylpyrrolidone provided long-term stability, allowing chips to be stored for up to 1 month with good viability. Detection of a recombinant BoNTA light chain was then carried out at 37 °C and a sub-lethal dose was detected in 2 hours.     

The effect of 1-(4,5-dihydro-1H-imidazol-2-yl) isoquinoline on monoamine release and turnover in the rat frontal cortex

Imidazoline-(2) binding sites (I(2)-BS) are widely distributed in rat brain and our studies have shown that drugs selective for these sites regulate central extrasynaptic monoamine concentrations. Radioligand binding studies have recently shown that BU98008 (1-[4,5-dihydro-1H-imidazol-2-yl] isoquinoline) displays high affinity at I(2)-binding sites. The aim of this study was set to assess the pharmacological actions of BU98008 in a functional in vivo model using the technique of in vivo brain microdialysis. Systemic injection of 20 mg/kg BU98008 produced an 85% rise in extracellular noradrenaline levels compared with basal values in the rat frontal cortex. Further experiments demonstrated that peripheral administration of 10 and 20 mg/kg BU98008 elicited a transient 25% elevation in dopamine overflow compared with basal values and simultaneously produced an 18% decrease in extracellular DOPAC (3-4-dihydroxyphenylacetic acid) levels compared to basal values. In addition, BU98008 did not appear to affect serotonergic neurotransmission in the frontal cortex. In conclusion, the present study demonstrates that BU98008 shares some functional similarities with known selective I(2)-BS ligands.     

Dynorphin A (1-17), Met-enkephalin-Arg6-Phe7 and substance P (1-11) levels in the brain of mice with different levels of ethanol consumption

Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 116, N ^o 8, pp. 177–178, August, 1993     

Interactions between vascular endothelial growth factor and neuroglobin

Vascular endothelial growth factor (VEGF) and neuroglobin (Ngb) participate in neuronal responses to hypoxia and ischemia, but the relationship between their effects, if any, is unknown. To address this issue, we measured Ngb levels in VEGF-treated mouse cerebrocortical cultures and VEGF levels in cerebrocortical cultures from Ngb-overexpressing transgenic mice. VEGF stimulated Ngb expression in a VEGFR2/Flk1 receptor-dependent manner, whereas Ngb overexpression suppressed expression of VEGF. These findings provide further insight into hypoxia-stimulated neuronal signaling pathways.     

Sunlight, the vitamin D endocrine system, and their relationships with gynaecologic cancer

Vitamin D has classically been considered an important nutrient, but modern scientific evidence points out that it has a new and more critical role as ubiquitous hormone at the centre of a complex endocrine, paracrine, and autocrine system involved in maintaining general health. Vitamin D is found in small quantities in food; however, it is also produced by the skin when exposed to certain intensities of ultraviolet light. Substantial epidemiological and clinical data suggest a link between low levels of vitamin D and an increased risk of a number of female specific cancers. Different types of cancer cells present vitamin D receptors and the enzymatic system involved in both vitamin D synthesis and inhibition. Sustained low levels of its precursor are responsible for alterations in vitamin D tissue and cell production and metabolism. The active form of vitamin D, 1,25(OH)2D3, can induce differentiation, inhibit proliferation, and modulate immune responsiveness of breast and a wide variety of female genital cell types. Vitamin D effects have been observed on expression of cell cycle regulators, growth factors and their receptors, apoptotic machinery, metastatic potential, and angiogenesis; all of which have some effect on hyperproliferative conditions. However, vitamin D blood levels may not be representative of the local metabolic alterations during carcinogenesis. Clinical studies support the recommendation to increase vitamin D levels to a normal range in order to prevent the tissue disorders related to hypovitaminosis D which are thought to be involved in the initiation and progression of cancer.     

Adenosine A2A receptors in ventral striatum, hypothalamus and nociceptive circuitry implications for drug addiction, sleep and pain

Adenosine A2A receptors localized in the dorsal striatum are considered as a new target for the development of antiparkinsonian drugs. Co-administration of A2A receptor antagonists has shown a significant improvement of the effects of l-DOPA. The present review emphasizes the possible application of A2A receptor antagonists in pathological conditions other than parkinsonism, including drug addiction, sleep disorders and pain. In addition to the dorsal striatum, the ventral striatum (nucleus accumbens) contains a high density of A2A receptors, which presynaptically and postsynaptically regulate glutamatergic transmission in the cortical glutamatergic projections to the nucleus accumbens. It is currently believed that molecular adaptations of the cortico-accumbens glutamatergic synapses are involved in compulsive drug seeking and relapse. Here we review recent experimental evidence suggesting that A2A antagonists could become new therapeutic agents for drug addiction. Morphological and functional studies have identified lower levels of A2A receptors in brain areas other than the striatum, such as the ventrolateral preoptic area of the hypothalamus, where adenosine plays an important role in sleep regulation. Although initially believed to be mostly dependent on A1 receptors, here we review recent studies that demonstrate that the somnogenic effects of adenosine are largely mediated by hypothalamic A2A receptors. A2A)receptor antagonists could therefore be considered as a possible treatment for narcolepsy and other sleep-related disorders. Finally, nociception is another adenosine-regulated neural function previously thought to mostly involve A1 receptors. Although there is some conflicting literature on the effects of agonists and antagonists, which may partly be due to the lack of selectivity of available drugs, the studies in A2A receptor knockout mice suggest that A2A receptor antagonists might have some therapeutic potential in pain states, in particular where high intensity stimuli are prevalent.     

Vitamin D and its implications for musculoskeletal health in women: an update

Vitamin D is a hormone that controls phosphorus, calcium, and bone metabolism and neuromuscular function. Vitamin D synthesis is a process in which the skin, liver, and kidney are sequentially involved. The vitamin D pool is completed by the amount taken with food and supplements. Vitamin D deficiency causes osteopenia, precipitates and exacerbates osteoporosis, causes a painful disease, osteomalacia, and increases muscle weakness, which worsens the risk of falls and fractures. A high prevalence of vitamin D insufficiency exists in the apparently healthy population, osteoporotic patients, and patients with prior fractures. Factors contributing to low vitamin D levels include low sunlight exposure, decreased skin synthesis and intestinal absorption, and inadequate diet. The simplest way to correct hypovitaminosis is adequate nutrition and supplements. However, few patients with osteoporosis and/or fractures, receive adequate supplements. Vitamin D insufficiency may alter the regulatory mechanisms of parathyroid hormone and may induce a secondary hyperparathyroidism that increases the risk of osteoporosis and fractures, although the necessary degree of this is not established. Monitoring of serum 25-hydroxyvitamin D levels is the only way to assess vitamin D status. The ideal healthy blood levels of 25-hydroxyvitamin D are controversial, although a range from 30 to 60ng/mL is widely accepted. The role of vitamin D supplementation is to provide humans with the nutrient in an amount closer to the biological norm for our species. This amount of vitamin D results in optimal function of many aspects of health, including balance and muscle strength, thus reducing the risk of fracture beyond what is possible via the quality and quantity of bone itself.     

Muscarinic cholinergic receptor subtypes in cerebral cortex of Fisher 344 rats: a light microscope autoradiography study of age-related changes

The density and localization of muscarinic cholinergic M1-M5 receptor subtypes was investigated in frontal and occipital cortex of male Fisher 344 rats aged 6 months (young-adult), 15 months (mature) and 22 months (senescent) by combined kinetic and equilibrium binding and light microscope autoradiography. In 6-month-old rats, the rank order density of muscarinic cholinergic receptor subtypes was M1>M2>M4>M3>M5 both in frontal and occipital cortex. A not homogeneous distribution of different receptor subtypes throughout cerebrocortical layers of frontal or occipital cortex was found. In frontal cortex silver grains corresponding to the M1 and M2 receptor subtypes were decreased in 15- and 22-month-old groups. The M3 receptor density was remarkably and moderately decreased in layers II/III and V, respectively, of rats aged 15 and 22 months. A reduced M4 receptor density was observed in layer I and to a lesser extent in layer V of mature and senescent rats, whereas no age-related changes of M5 receptor were found. In occipital cortex a diminution of M1 receptor was observed in layers II/III and V of mature and senescent rats. The M2 receptor expression decreased in layer I of 15- and 22-month-old senescent rats, whereas M3-M5 receptors were unchanged with exception of a slight decrease of the M4 receptor in layer IV and of M5 receptor in layers II/III. These findings indicate a different sensitivity to aging of muscarinic receptor subtypes located in various cerebrocortical layers. This may account for the difficulty in obtaining relevant results in manipulating cholinoceptors to counter age-related impairment of cholinergic system.     

Activity of growth hormone-releasing peptide-2 in cultured human pituitary adenoma cells and its interaction with growth hormone-releasing hormone and somatostatin

Experiments on primary cultures of human pituitary adenoma cells producing growth hormone (GH) or GH and prolactin showed that similarly to GH-releasing hormone (GHRH) synthetic hexapeptide GH-releasing peptide-2 (GHRP) directly enhance secretion of GH but not of prolactin by human pituitary cells. The effect of various doses of GHRP and GHRH applied in combination was additive or slightly synergistic in nature. Somatostatin inhibits secretion of GH induced by GHRP, GHRH, or their combination. A dissociation is found between the inhibitory effects of somatostatin on basal and stimulated GH secretion. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 125, No. 2, pp. 207–212, February, 1998     

The osteocyte--a novel endocrine regulator of body phosphate homeostasis

Although osteocytes are the most abundant cell type in bone, much of their biology remains enigmatic. They are known to transduce mechanical stress into signals that initiate local bone remodeling, and are targets for systemic and local endocrine signals that affect bone architecture and mineral homeostasis. However, recent data reveal that osteocytes themselves act as endocrine cells that synthesize fibroblast growth factor 23 (FGF23) and several other phosphatonins, shown to underpin the systemic regulation of phosphate homeostasis. This review will synthesize the emerging discoveries concerning the osteocytic endocrine role in phosphate homeostasis through the biology and pathophysiology of these phosphatonins. We also suggest future research paths that might resolve existing uncertainties, and look ahead at how greater understanding might improve the management of clinical disorders of phosphate homeostasis.     

Adenosine A2A receptors and brain injury: broad spectrum of neuroprotection, multifaceted actions and "fine tuning" modulation

This review summarizes recent developments that have contributed to understand how adenosine receptors, particularly A2A receptors, modulate brain injury in various animal models of neurological disorders, including Parkinson's disease (PD), stroke, Huntington's disease (HD), multiple sclerosis, Alzheimer's disease (AD) and HIV-associated dementia. It is clear that extracellular adenosine acting at adenosine receptors influences the functional outcome in a broad spectrum of brain injuries, indicating that A2A Rs may modulate some general cellular processes to affect neuronal cells death. Pharmacological, neurochemical and molecular/genetic approaches to the complex actions of A2A receptors in different cellular elements suggest that A2A receptor activation can be detrimental or protective after brain insults, depending on the nature of brain injury and associated pathological conditions. An interesting concept that emerges from these studies is A2A R's ability to fine tune neuronal and glial functions to produce neuroprotective effects. While the data presented here clearly highlight the complexity of using adenosinergic agents therapeutically in PD and other neurodegenerative disorders and point out many areas for further inquiry, they also confirm that adenosine receptor ligands, particularly A2A receptor ligands, have many promising characteristics that encourage the pursuit of their therapeutic potential.     

Role of adenosine A2A receptors in parkinsonian motor impairment and l-DOPA-induced motor complications

Adenosine A2A receptors have a unique cellular and regional distribution in the basal ganglia, being particularly concentrated in areas richly innervated by dopamine such as the caudate-putamen and the globus pallidus. Adenosine A2A receptors are selectively located on striatopallidal neurons and are capable of forming functional heteromeric complexes with dopamine D2 and metabotropic glutamate mGlu5 receptors. Based on the unique cellular and regional distribution of this receptor and in line with data showing that A2A receptor antagonists improve motor symptoms in animal models of Parkinson's disease (PD) and in initial clinical trials, A2A receptor antagonists have emerged as an attractive non-dopaminergic target to improve the motor deficits that characterize PD. Experimental data have also shown that A2A receptor antagonists do not induce neuroplasticity phenomena that complicate long-term dopaminergic treatments. The present review provides an updated summary of results reported in the literature concerning the biochemical characteristics and basal ganglia distribution of A2A receptors. We subsequently aim to examine the effects of adenosine A2A antagonists in rodent and primate models of PD and of l-DOPA-induced dyskinesia. Finally, concluding remarks are made on post-mortem human brains and on the translation of adenosine A2A receptor antagonists in the treatment of PD.     

The energy hypothesis of sleep revisited

One of the proposed functions of sleep is to replenish energy stores in the brain that have been depleted during wakefulness. Benington and Heller formulated a version of the energy hypothesis of sleep in terms of the metabolites adenosine and glycogen. They postulated that during wakefulness, adenosine increases and astrocytic glycogen decreases reflecting the increased energetic demand of wakefulness. We review recent studies on adenosine and glycogen stimulated by this hypothesis. We also discuss other evidence that wakefulness is an energetic challenge to the brain including the unfolded protein response, the electron transport chain, NPAS2, AMP-activated protein kinase, the astrocyte-neuron lactate shuttle, production of reactive oxygen species and uncoupling proteins. We believe the available evidence supports the notion that wakefulness is an energetic challenge to the brain, and that sleep restores energy balance in the brain, although the mechanisms by which this is accomplished are considerably more complex than envisaged by Benington and Heller.