Anandamide produced by Ca2+-insensitive enzymes induces excitation in primary sensory neurons

The endogenous lipid agent N-arachidonoylethanolamine (anandamide), among other effects, has been shown to be involved in nociceptive processing both in the central and peripheral nervous systems. Anandamide is thought to be synthesised by several enzymatic pathways both in a Ca²⁺-sensitive and Ca²⁺-insensitive manner, and rat primary sensory neurons produce anandamide. Here, we show for the first time, that cultured rat primary sensory neurons express at least four of the five known Ca²⁺-insensitive enzymes implicated in the synthesis of anandamide, and that application of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-arachidonoyl, the common substrate of the anandamide-synthesising pathways, results in anandamide production which is not changed by the removal of extracellular Ca²⁺. We also show that anandamide, which has been synthesised in primary sensory neurons following the application of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-arachidonoyl induces a transient receptor potential vanilloid type 1 ion channel-mediated excitatory effect that is not inhibited by concomitant activation of the cannabinoid type 1 receptor. Finally, we show that sub-populations of transient receptor potential vanilloid type 1 ion channel-expressing primary sensory neurons also express some of the putative Ca²⁺-insensitive anandamide-synthesising enzymes. Together, these findings indicate that anandamide synthesised by primary sensory neuron via a Ca²⁺-insensitive manner has an excitatory rather than an inhibitory role in primary sensory neurons and that excitation is mediated predominantly through autocrine signalling. Regulation of the activity of the Ca²⁺-insensitive anandamide-synthesising enzymes in these neurons may be capable of regulating the activity of these cells, with potential relevance to controlling nociceptive processing.     

Recommendations for reporting results of diagnostic genetic testing (biochemical,cytogenetic and molecular genetic)

Genetic test results can have considerable importance for patients, their parents and more remote family members. Clinical therapy and surveillance, reproductive decisions and genetic diagnostics in family members, including prenatal diagnosis, are based on these results. The genetic test report should therefore provide a clear, concise, accurate, fully interpretative and authoritative answer to the clinical question. The need for harmonizing reporting practice of genetic tests has been recognised by the External Quality Assessment (EQA), providers and laboratories. The ESHG Genetic Services Quality Committee has produced reporting guidelines for the genetic disciplines (biochemical, cytogenetic and molecular genetic). These guidelines give assistance on report content, including the interpretation of results. Selected examples of genetic test reports for all three disciplines are provided in an annexe.Diagnostic genetic testing is an extremely rapidly expanding area encompassing a broad range of laboratory investigations to analyse chromosomes (from classical karyotype to molecular cytogenetics), nucleic acids (DNA, RNA), proteins and metabolites used to detect heritable or somatic mutations, genotypes or phenotypes related to disease and health. Genetic testing requires particular consideration in that it is usually performed only once in a patient''s lifetime, and the results may have considerable importance for lifetime decisions not only for the individuals being tested but also for children and family. Interpreting and reporting variation in germline chromosomes, DNA sequences or their products is a heavy clinical responsibility for prediction of susceptibility to disease, patient diagnosis, prognosis, counselling, treatment or family planning. Providing a set of reporting frameworks that can be customised for different testing contexts but share some common principles could be beneficial to the practice of a number of laboratories, including non-OECD members and/or laboratories that do not participate in External Quality Assessments (EQA), and to laboratories with blurred boundaries between research and genetic testing services.Although several guidelines already exist for reporting the results of genetic testing,^1,2,3 these focus on molecular genetic testing and do not cover the other two branches of laboratory genetics, namely biochemical genetics and cytogenetics. Based on recent surveys of EQA results presented by some European EQA providers and the request from genetic laboratories for comprehensive reporting guidelines, it was considered that a unifying attempt to harmonise the reporting practice of genetic tests in Europe and neighbouring countries would be welcome.     

Electrical and optical study of nerve impulse-evoked ATP-induced, P2X-receptor-mediated sympathetic neurotransmission at single smooth muscle cells in mouse isolated VAS deferens

Simultaneous electrophysiology and confocal microscopy were used to investigate purinergic neurotransmission at single smooth muscle cells (SMCs) in mouse isolated vas deferens, and to explore the relationship between two high-resolution P2X-receptor-mediated measures of per pulse ATP release: transient peaks in the first time derivative of the rising phase of excitatory junction potentials (EJPs) recorded in single SMCs ('discrete events'; DEs) and neuroeffector Ca(2+) transients (NCTs) in the impaled SMCs. This study shows that discrete events represent neurotransmitter release onto the impaled cell. First, the median amplitude of the first derivative of the EJP was larger when there was a coincident NCT in the impaled cell, compared with instances when no coincident NCT occurred. Second, the time-to-peak amplitude of the first derivative was shorter if there was a coincident NCT in the impaled cell, compared with when no coincident NCT was observed within the field. Surprisingly, first derivative amplitude increased with the distance (of the corresponding NCT) from the microelectrode. The microelectrode did not locally inhibit the functional quantal size as there was no effect of distance on the normalized NCT amplitude. When the significant effect of distance (between the microelectrode and NCTs) on the first derivative amplitude was removed, there was no correlation between the unstandardized residual (of distance vs. first derivative amplitude) and NCT amplitude. The absence of a correlation between DE and NCT amplitudes suggests that the NCT amplitude is a poor measure of quantal size. The usefulness of NCTs hence lies primarily in locating neurotransmitter release and measuring changes in local release probability.     

Refrigerated storage of lyophilized and rehydrated, lyophilized human red cells

Human red cells (RBCs) were collected in CPDA-1 and then freeze-dried in lyoprotective solution. The lyophilized RBCs were then stored at -20 degrees C for 7 days. At the end of the storage period, the lyophilized RBCs were rehydrated and washed in dextrose saline. The washed, reconstituted, lyophilized RBCs were resuspended in final wash solutions of ADSOL, CPDA-1, or a special additive solution containing glucose, citrate, phosphate, adenine, and mannitol, and then they were stored at 4 degrees C for an additional 7 days. The main purpose of this study was to determine whether human RBCs can be lyophilized in such a manner that normal metabolic, rheologic, and cellular properties are maintained during rehydration and subsequent storage in standard blood bank preservative solutions. Our results show that reconstituted, lyophilized RBCs maintained levels of ATP, 2,3 DPG, lactate, and cellular properties that are equal to or better than those in control nonlyophilized RBCs stored for a comparable period in CPDA-1. Reconstituted, lyophilized RBCs stored at 4 degrees C after rehydration also show better maintenance of ATP, 2,3 DPG, and lactate than do control RBCs stored in the same preservative solutions for comparable periods.     

PCR-positivity in harvested bone marrow predicts relapse after transplantation with autologous purged bone marrow in children in second remission of precursor B-cell acute leukaemia

Purging of autologous bone marrow (BM) grafts of children in second remission after a relapse of precursor B acute lymphoblastic leukaemia (ALL) in the BM has been carried out in our laboratory since 1987, initially by complement mediated cell lysis. This protocol was extended by performing an immunorosette depletion before lysis with complement. The aim of the present study was to assess by polymerase chain reaction the presence of residual leukaemic cells in the BM grafts before and after purging. The results were then correlated to clinical outcome. In 24/28 patients a PCR product was obtained by amplification of IgH and/or TcR junctional regions. BM before purging was available for analysis in 13 patients. We found that leukaemic cells could be detected in 8/13 (62%) of these grafts before purging . All these eight patients experienced a relapse, regardless of whether the purging procedure had been successful (defined as achievement of PCR-negativity) or not. In contrast, none of the five patients with PCR-negative grafts before purging relapsed ( P  = 0.0008). One patient died due to transplant-related toxicity. Of the remaining 23 patients, nine patients received a PCR-positive BM graft after purging. All these nine patients experienced a relapse as compared to 6/14 whose BM was PCR-negative after purging ( P  = 0.0072). Two of eight PCR-positive BM grafts could be purged to PCR-negativity. Thus, improvements both in treatment of leukaemia and in purging efficacy are still needed.     

Stress-induced suppression of the gonadotropin-releasing hormone pulse generator in the female rat: a novel neural action for calcitonin gene-related peptide

In addition to its role as a potent vasodilator, calcitonin gene-related peptide (CGRP) is centrally involved in a variety of stress responses, including activation of the hypothalamo-pituitary-adrenocortical axis. It is well known that stress suppresses the activity of the hypothalamic GnRH pulse generator, the central regulator of LH and FSH pulses, resulting in reproductive dysfunction. The aim of this study was to test the hypothesis that CGRP has a critical role in mediating stress-induced suppression of pulsatile LH secretion in the rat. Ovariectomized rats were implanted with intracerebroventricular and iv cannulae. Central administration of CGRP (75 pmol-1.2 nmol) into the lateral cerebral ventricle resulted in a profound, dose-dependent suppression of LH pulses, which was reversed by a CGRP receptor antagonist (CGRP(8-37),1 nmol). Although the site of action of CGRP remains to be established, the induction of c-Fos expression in the preoptic area and hypothalamic paraventricular nucleus might suggest an involvement of these brain regions. Intravenous administration of CGRP did not affect LH pulses. Coadministration (intracerebroventricular) of CGRP (400 pmol) with a CRH antagonist (alpha-helical CRF(9-41), 26 nmol) partly blocked the CGRP-induced suppression of LH pulses. Furthermore, CGRP(8-37) (1 nmol) completely blocked hypoglycemic stress-induced suppression of LH pulses. These results suggest that the suppression of pulsatile LH secretion by central administration of CGRP may be mediated in part by CRH, and that CGRP may play a pivotal role in the normal physiological response of stress-induced suppression of the hypothalamic GnRH pulse generator, and hence the reproductive system.     

维思通对女性精神病人血清催乳素及月经周期的影响

目的　探讨维思通对女性精神病人血清催乳素 (PRL)及月经周期的影响 ,同时也观察了这一副反应对病人的情绪影响。方法　对 5 6例服用维思通的首发精神分裂症女性病人进行连续 12周的治疗观察 ,并测定用药前和用药后第 4周、12周的PRL数值 ;以及观察 3次月经周期变化。在疗前及 12周治疗结束后评定BPRS、TESS。结果　第 4周内病人PRL变化不明显 (0 0 5

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