Relaxin-3/RXFP3 signalling in mouse hypothalamus: no effect of RXFP3 activation on corticosterone, despite reduced presynaptic excitatory input onto paraventricular CRH neurons in vitro.

Relaxin-3/RXFP3 signalling is proposed to be involved in the neuromodulatory control of arousal- and stress-related neural circuits. Furthermore, previous studies in rats have led to the proposal that relaxin-3/RXFP3 signalling is associated with activation of the hypothalamic-pituitary-adrenal axis, but direct evidence for RXFP3-related actions on the activity of hypothalamic corticotropin-releasing hormone (CRH) neurons is lacking. In this study, we investigated characteristics of the relaxin-3/RXFP3 system in mouse hypothalamus. Administration of an RXFP3 agonist (RXFP3-A2) intra-cerebroventricularly or directly into the paraventricular nucleus of hypothalamus (PVN) of C57BL/6J mice did not alter corticosterone levels. Similarly, there were no differences between serum corticosterone levels in Rxfp3 knockout (C57BL/6JRXFP3TM1) and wild-type mice at baseline and after stress, despite detection of the predicted stress-induced increases in serum corticosterone. We examined the nature of the relaxin-3 innervation of PVN in wild-type mice and in Crh-IRES-Cre;Ai14 mice that co-express the tdTomato fluorophore in CRH neurons, identifying abundant relaxin-3 fibres in the peri-PVN region, but only sparse fibres associated with densely packed CRH neurons. In whole-cell voltage-clamp recordings of tdTomato-positive CRH neurons in these mice, we observed a reduction in sEPSC frequency following local application of RXFP3-A2, consistent with an activation of RXFP3 on presynaptic glutamatergic afferents in the PVN region. These studies clarify the relationship between relaxin-3/RXFP3 inputs and CRH neurons in mouse PVN, with implications for the interpretation of current and previous in vivo studies and future investigations of this stress-related signalling network in normal and transgenic mice, under normal and pathological conditions.

Validation of the accuracy of needle placement as used in diagnostic local analgesia of the maxillary nerve for investigation of trigeminally mediated headshaking in horses.

Diagnostic local anaesthesia of the maxillary nerve is a valuable aid in the diagnosis of trigeminally mediated headshaking in horses. Our objective is to validate the accuracy of needle placement in this procedure and to identify any correlation between accuracy of the technique and operator experience. Using a small volume of contrast medium, the procedure was performed bilaterally on 30 horse cadaver heads by three groups with different levels of experience with the technique. The location of deposition was then identified using computed tomography (CT). Contrast medium was deposited around the target site in 53.3% (32/60) of injections. An experienced operator succeeded in deposition around the target area significantly (p<0.05) more often (80%, 16/20) than did the less and non-experienced performers (40%, 16/40). A negative response to diagnostic local anaesthesia of the maxillary nerve does not disprove facial dysaesthesia as the cause of headshaking in that horse as a false negative response could arise due to failure to deposit local anaesthetic around the target area. Increased experience in performing the procedure decreases the probability of false negative results.

Identification of a calcitonin-like diuretic hormone that functions as an intrinsic modulator of the American lobster, Homarus americanus, cardiac neuromuscular system.

In insects, a family of peptides with sequence homology to the vertebrate calcitonins has been implicated in the control of diuresis, a process that includes mixing of the hemolymph. Here, we show that a member of the insect calcitonin-like diuretic hormone (CLDH) family is present in the American lobster, Homarus americanus, serving, at least in part, as a powerful modulator of cardiac output. Specifically, during an ongoing EST project, a transcript encoding a putative H. americanus CLDH precursor was identified; a full-length cDNA was subsequently cloned. In silico analyses of the deduced prepro-hormone predicted the mature structure of the encoded CLDH to be GLDLGLGRGFSGSQAAKHLMGLAAANFAGGPamide (Homam-CLDH), which is identical to a known Tribolium castaneum peptide. RT-PCR tissue profiling suggests that Homam-CLDH is broadly distributed within the lobster nervous system, including the cardiac ganglion (CG), which controls the movement of the neurogenic heart. RT-PCR analysis conducted on pacemaker neuron- and motor neuron-specific cDNAs suggests that the motor neurons are the source of the CLDH message in the CG. Perfusion of Homam-CLDH through the isolated lobster heart produced dose-dependent increases in both contraction frequency and amplitude and a dose-dependent decrease in contraction duration, with threshold concentrations for all parameters in the range 10(-11) to 10(-10) mol l(-1) or less, among the lowest for any peptide on this system. This report is the first documentation of a decapod CLDH, the first demonstration of CLDH bioactivity outside the Insecta, and the first detection of an intrinsic neuropeptide transcript in the crustacean CG.

The peptide hormone pQDLDHVFLRFamide (crustacean myosuppressin) modulates the Homarus americanus cardiac neuromuscular system at multiple sites.

pQDLDHVFLRFamide is a highly conserved crustacean neuropeptide with a structure that places it within the myosuppressin subfamily of the FMRFamide-like peptides. Despite its apparent ubiquitous conservation in decapod crustaceans, the paracrine and/or endocrine roles played by pQDLDHVFLRFamide remain largely unknown. We have examined the actions of this peptide on the cardiac neuromuscular system of the American lobster Homarus americanus using four preparations: the intact animal, the heart in vitro, the isolated cardiac ganglion (CG), and a stimulated heart muscle preparation. In the intact animal, injection of myosuppressin caused a decrease in heartbeat frequency. Perfusion of the in vitro heart with pQDLDHVFLRFamide elicited a decrease in the frequency and an increase in the amplitude of heart contractions. In the isolated CG, myosuppressin induced a hyperpolarization of the resting membrane potential of cardiac motor neurons and a decrease in the cycle frequency of their bursting. In the stimulated heart muscle preparation, pQDLDHVFLRFamide increased the amplitude of the induced contractions, suggesting that myosuppressin modulates not only the CG, but also peripheral sites. For at least the in vitro heart and the isolated CG, the effects of myosuppressin were dose-dependent (10(-9) to 10(-6) mol l(-1) tested), with threshold concentrations (10(-8)-10(-7) mol l(-1)) consistent with the peptide serving as a circulating hormone. Although cycle frequency, a parameter directly determined by the CG, consistently decreased when pQDLDHVFLRFamide was applied to all preparation types, the magnitudes of this decrease differed, suggesting the possibility that, because myosuppressin modulates the CG and the periphery, it also alters peripheral feedback to the CG.

Phytostabilization of gold mine tailings from New Zealand. Part 2: Experimental evaluation of arsenic mobilization during revegetation.

Revegetation of mine tailings usually requires amendments of phosphorus. However, phosphate addition can mobilize arsenic (As) from the tailings. A 5-mo lysimeter field trial was conducted to quantify As mobilization in gold mine tailings, in association with different P amendment products and different plant species (barley Hordeum vulgare, blue lupin Lupinus angustifolius, rye corn Secale cereale) necessary for short-term revegetation of mine tailings. A simultaneous laboratory experiment was run to examine As mobilization in 1-cm-deep tailings in relation to different P amendment rates. The experimental results showed that the amount of As leached was proportional to the amount of P added. In the larger scale lysimeters, P amendment of < 3 g m(-2) caused As leaching of 0.5 mg L(-1) from unplanted lysimeters and up to 0.9 mg L(-1) on average in planted lysimeters. Variable species-amendment combinations produced differences in the amount of As leached and uptaken. Leachates and uptakes were higher with an organic fertilizer amendment than Superphosphate, particularly in combination with barley. Arsenic accumulated in plant biomass to 126 mg kg(-1) in shoots and 469 mg kg(-1) in roots.

Acupuncture of chronic headache disorders in primary care: randomised controlled trial and economic analysis.

OBJECTIVES: To determine the effects of a policy of using acupuncture, compared with a policy of avoiding acupuncture, on headache in primary care patients with chronic headache disorders. The effects of acupuncture on medication use, quality of life, resource use and days off sick in this population and the cost-effectiveness of acupuncture were also examined. DESIGN: Randomised, controlled trial. SETTING: General practices in England and Wales. PARTICIPANTS: The study included 401 patients with chronic headache disorder, predominantly migraine. INTERVENTIONS: Patients were randomly allocated to receive up to 12 acupuncture treatments over 3 months or to a control intervention offering usual care. MAIN OUTCOME MEASURES: Outcome measures included headache score; assessment of Short Form 36 (SF-36) health status and use of medication at baseline, 3 months and 12 months; assessment of use of resources every 3 months; and assessment of incremental cost per quality-adjusted life-year (QALY) gained. RESULTS: Headache score at 12 months, the primary end-point, was lower in the acupuncture group than in controls. The adjusted difference between means was 4.6. This result was robust to sensitivity analysis incorporating imputation for missing data. Patients in the acupuncture group experienced the equivalent of 22 fewer days of headache per year. SF-36 data favoured acupuncture, although differences reached significance only for physical role functioning, energy and change in health. Compared with controls, patients randomised to acupuncture used 15% less medication, made 25% fewer visits to GPs and took 15% fewer days off sick. Total costs during the 1-year period of the study were on average higher for the acupuncture group than for controls because of the acupuncture practitioners' costs. The mean health gain from acupuncture during the year of the trial was 0.021 QALYs, leading to a base-case estimate of GBP9180 per QALY gained. This result was robust to sensitivity analysis. Cost per QALY dropped substantially when the analysis incorporated likely QALY differences for the years after the trial. CONCLUSIONS: The study suggests that acupuncture leads to persisting, clinically relevant benefits for primary care patients with chronic headache, particularly migraine. It is relatively cost-effective compared with a number of other interventions provided by the NHS. Further studies could examine the duration of acupuncture effects beyond 1 year and the relative benefit to patients with migraine with compared to tension-type headache. Trials are also warranted examining the effectiveness and cost-effectiveness of acupuncture in patients with headache receiving more aggressive pharmacological management.

Hypophosphatemia and the development of rickets in osteopetrotic (op/op) mice.

Our previous work has shown that op/op mice hyperabsorb dietary calcium in the vitamin D-deficient state and shunt that calcium into bone. Under these conditions, the op/op mice are hypocalcemic. The purpose of this study was to examine calcium metabolism and bone mineralization in vitamin D-deficient op/op mice. First, the op/op mice and their normal littermates were placed on a vitamin D-deficient, low phosphorus diet to limit bone mineralization. Under these circumstances, op/op mice survived, even when calcium was also removed from the diet. If the diet contained phosphate, op/op mice died from hypocalcemic tetany when calcium was also removed from the diet. Furthermore, serum calcium levels became similar to wild type in the op/op mice administered the vitamin D-deficient, low phosphorus diet, and op/op mice were able to increase serum calcium in response to 1,25-dihydroxyvitamin D3. The op/op mice developed rickets when their serum phosphorus level was too low to support bone mineralization. The op/op mice became hypophosphatemic on regimens in which normal mice were able to maintain normal serum phosphorus levels. It appears that the op/op mouse simply requires a higher dietary calcium and phosphorus level to prevent rickets and hypocalcemic tetany since the bone is not available as a source of these minerals. However, the ability of the op/op mouse to mineralize bone at low serum calcium and phosphorus levels remains unexplained.

The effect of pantothenate deficiency in mice on their metabolic response to fast and exercise.

The changes in fuel metabolism during fast and exercise were compared to the tissue total CoA levels in mice maintained on pantothenate-deficient and pantothenate-supplemented (control) diets. In nonexercised mice maintained on a pantothenate-deficient diet for 65 to 105 days, the total CoA levels of many tissues were significantly lower than in controls (liver 18%, kidney 23%, spleen 21%, heart 38%, and leg skeletal muscle 66%). However, no differences in total CoA levels in brain or epididymal fat pads were observed. During a 48-hour fast, the total CoA levels increased in the heart and liver of both pantothenate-deficient and control mice (heart 32 and 19%, respectively; liver 39 and 45%, respectively), but the level of total CoA remained lower in the deficient mice. Liver glycogen levels were 17% lower in deficient mice than in controls and liver ketone bodies were 17% higher in pantothenate deficient mice than in controls. Separate groups of mice on deficient and supplemented diets were trained to run to exhaustion. Compared to trained mice on pantothenate-supplemented diets, the trained pantothenate-deficient mice had lower running times until exhaustion, lower body weights, lower liver and muscle glycogen content (even after rest), and elevated liver ketone bodies both during rest and after running. In summary, the pantothenate-deficient mice were unable to maintain normal glycogen stores, but had a normal ketogenic response to fast and exercise in spite of the lower levels of liver total CoA.

Diaphragmatic breathing training and walking performance in chronic airways obstruction.

Eight patients with chronic obstructive bronchitis and moderate disability entered a pilot study of the effects of controlled diaphragmatic breathing. They received three weeks of placebo physiotherapy (shoulder exercises) followed by three weeks of instruction on controlled diaphragmatic breathing. No beneficial effects were observed on exercise performance or the perceived strain of exercise.

Effect of lead ingestion on functions of vitamin D and its metabolites.

A study of the effect of ingestion of lead on the metabolism and function of vitamin D was carried out in rats fed diets varying in calcium and phosphorus content. The ingestion of 0.82% lead as lead acetate suppressed plasma levels of 1,25-dihydroxycholecalciferol in rats fed either a low phosphorus or a low calcium diet while it had no effect on this parameter in rats fet either a high calcium diet or a normal phosphorus diet. Most important, the ingestion of lead totally blocked the intestinal calcium transport response to cholecalciferol, 25-hydroxycholecalciferol and 1,25-dihydroxycholecalciferol. On the other hand, the ingestion of lead acetate had no influence on the mobilization of calcium from bone, the elevation of serum inorganic phosphorus and in the mineralization of rachitic bone in the same animals. Thus by the feeding of 0.82% lead on the intestinal responses to vitamin D and its metabolites was greatest in animals fed a low calcium or a low phosphorus diet, it was present with all diets tested.

Psychogenic purpura in a man.

A 53-year-old man with chronic back and leg pain developed recurrent painful ecchymoses after lumbar laminectomy. No hematologic abnormality could be detected, but an ecchymosis developed after subcutaneous injection of his blood into the region of pain. A detailed study of the psychological setting of the illness and his personality revealed this to be an example of psychogenic purpura. This is the third report of the syndrome in a male.