Development of SHIRPA to characterise the phenotype of gene-targeted mice.
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Animals↗
Biomedical subjects
D N Jones
Publications and source records attributed to D N Jones.
Evidence that orexin-A-evoked grooming in the rat is mediated by orexin-1 (OX1) receptors, with downstream 5-HT2C receptor involvement.
RATIONALE: Orexins A and B have recently been discovered and shown to be derived from preproorexin, primarily expressed in the rat hypothalamus. Orexin-A has been ascribed a number of in vivo functions in the rat after intracerebroventricular (ICV) administration, including hyperphagia, neuroendocrine modulation and, most recently, evidence for a behavioural response characterised by an increase in grooming. OBJECTIVES: Here, we have investigated the orexin-receptor subtypes involved in the grooming response to orexin-A (3 microg, ICV) in the rat. METHODS: Male rats, habituated to clear Perspex behavioural observation boxes, were pretreated with antagonists with mixed selectivity for OX1, OX2, 5-HT2B and 5-HT2C receptor subtypes prior to the administration of orexin-A and the intense grooming response elicited by this peptide assessed. RESULTS: Pretreatment of rats with a mixed OX1/5-HT2B/2C receptor antagonist 1-(4-methylsulfanylphenyl)-3-quinolin-4-ylurea (SB-284422), revealed a significant, but incomplete, blockade of orexin-A-induced grooming. Despite the low potency of orexin-A at 5-HT2B and 5-HT2C receptors in vitro (pKi<5), studies were undertaken to determine whether downstream 5-HT2B or 5-HT2C receptors mediate in the grooming-elicited by orexin-A. Whilst the selective 5-HT2B receptor antagonist, SB-215505 (3 mg/kg, PO, 5-HT2B, pKi=8.58; OX1, pKB < 5.15) failed to effect orexin-A-induced grooming, the selective 5-HT2C receptor antagonist, SB-242084 (1 mg/kg, IP, 5-HT2C, pKi = 8.95; OX1, pKB < 5.1) potently antagonised the grooming response to this peptide. This suggested that the partial blockade of orexin-A-induced grooming obtained with SB-284422 might be attributable to its 5-HT2C and/or OX1 receptor blocking activity. However, complete blockade of orexin-A-induced grooming by the subsequently identified selective OX1 receptor antagonist 1-(2-methylbenzoxazol-6-yl)-3-[1,5]naphthyridin-4-yl urea hydrochloride, SB-334867-A (OX1, pKB = 7.4; OX2, pKB = 5.7), devoid of appreciable affinity for either 5-HT2B (pKi < 5.3) or 5-HT2C (pKi < 5.4) receptors, provides the first definitive evidence that a central behavioural effect of orexin-A (grooming) is mediated by OX1 receptors. CONCLUSIONS: This data suggests that orexin-A indirectly activates 5-HT2C receptors downstream from OX1 receptors to elicit grooming in the rat. The use of SB-334867-A in vivo will enable the role of OX,1 receptors within the rat central nervous system to be further characterised.
Animals↗
Effects of centrally administered orexin-B and orexin-A: a role for orexin-1 receptors in orexin-B-induced hyperactivity.
RATIONALE: Orexin-A and orexin-B are hypothalamic neuropeptides derived from a 130-amino acid precursor, prepro-orexin, and are potent agonists at both the orexin-1 (OX1) and orexin-2 (OX2) receptors. Orexin-A has been ascribed a number of in vivo functions in the rat after intracerebroventricular (ICV) administration, including hyperphagia, neuroendocrine modulation and a role in the regulation of sleep-wake function. The in vivo role of orexin-B is not as clear. OBJECTIVES: To investigate the behavioural, endocrine and neurochemical effects of orexin-B in in-vivo tests. In a number of experiments, these effects were compared with those of orexin-A. METHODS: Experiments were carried out in male, Sprague-Dawley rats with a guide cannula directed towards the lateral ventricle. The effects of orexin-B (ICV) upon grooming behaviour were compared with those of orexin-A. The effects of orexin-B upon the motor activity response to both novel and familiar environments were assessed in an automated activity monitor. Orexin-B was tested upon startle reactivity and body temperature. Further, plasma hormones and [DOPAC+ HVA]/[DA] and [5-HIAA]/[5-HT] ratios in six brain areas were measured 40 min post-orexin-B or orexin-A. RESULTS: The clearest behavioural response to orexin-B was increased motor activity in both novel and familiar environments. Orexin-B-induced hyperactivity was blocked by an OX1 receptor antagonist, SB-334867-A, implicating OX1 receptors in this behavioural response. In common with orexin-A, orexin-B reduced plasma prolactin and failed to influence startle reactivity. However, in contrast with orexin-A, orexin-B increased head grooming but failed to cause a robust whole body grooming response or increase plasma corticosterone levels. Further, orexin-B, but not orexin-A, increased plasma TSH and increased hypothalamic and striatal [5-HIAA]/[5-HT] ratios. CONCLUSIONS: The present study has demonstrated a number of behavioural, neuroendocrine and neurochemical effects of orexin-B that distinguish it from orexin-A. Further, we have demonstrated a role for OX1 receptors in the actions of orexin-B upon motor activity.
Animals↗
Central effects of urotensin-II following ICV administration in rats.
RATIONALE: Urotensin-II (U-II) has recently been identified as an agonist for the G-protein-coupled receptor, GPR14. Detection of both U-II and GPR14 mRNA in the brain and spinal cord is consistent with a role for U-II in the CNS. However, the effects of central administration of U-II in rodents have not been reported previously. OBJECTIVES: To determine the localisation of GPR14 mRNA in rat tissues and to investigate the behavioural and endocrine effects of human U-II (hU-II) following intracerebroventricular (ICV) administration in rats. METHODS: Experiments were carried out in male Sprague-Dawley rats. Expression of GPR14 mRNA in rat brain was determined by semi-quantitative RT-PCR. Effects of hU-II on general behaviours were assessed by an observer and the motor activity response was measured by an automated activity monitor. Plasma hormones and [DOPAC + HVA]/[DA] and [5-HIAA]/[5-HT] ratios in five brain areas were measured 20 min post-hU-II (ICV). RESULTS: GPR14 mRNA expression was found in whole brain tissue and in all CNS regions tested. GPR14 mRNA expression was also detected in the periphery; highest levels were found in the heart. Following ICV administration, hU-II (3-10 micrograms ICV) increased rearing and grooming, and increased motor activity in a familiar environment. Further, hU-II increased plasma prolactin and TSH but did not affect levels of corticosterone. hU-II had no effects on dopamine or 5-HT levels or their metabolites in the frontal cortex, hippocampus, hypothalamus, striatum and nucleus accumbens. CONCLUSIONS: These data provide further insight into the distribution of GPR14 mRNA within the CNS and show for the first time that hU-II causes marked behavioural and endocrine effects.
Animals↗
Increased responsiveness of dopamine to atypical, but not typical antipsychotics in the medial prefrontal cortex of rats reared in isolation.
Dopaminergic hypofunction in the medial prefrontal cortex (mPFC) has been associated with the aetiology of negative symptoms and cognitive dysfunction of schizophrenia, which are both alleviated by clozapine and other atypical antipsychotics such as olanzapine. In rodents, early life exposure to stressful experiences such as social isolation produces a spectrum of symptoms emerging in adult life, which can be restored by antipsychotic drugs. The present series of experiments sought to investigate the effect of clozapine (5-10 mg/kg s.c.), olanzapine (5 mg/kg s.c.), and haloperidol (0.5 mg/kg s.c.) on dopamine (DA) and amino acids in the prelimbic/infralimbic subregion of the mPFC in group- and isolation-reared rats. Rats reared in isolation showed significant and robust deficits in prepulse inhibition of the acoustic startle. In group-reared animals, both clozapine and olanzapine produced a significant increase in DA outflow in the mPFC. Isolation-reared rats showed a significant increase in responsiveness to both atypical antipsychotics compared with group-reared animals. In contrast, the administration of haloperidol failed to modify dialysate DA levels in mPFC in either group- or isolation-reared animals. The results also show a positive relationship between the potency of the tested antipsychotics to increase the release of DA in the mPFC and their respective affinities for 5-HT1A relative to DA D2 or D3 receptors. Finally, isolation-reared rats showed enhanced neurochemical responses to the highest dose of clozapine as indexed by alanine, aspartate, GABA, glutamine, glutamate, histidine, and tyrosine. The increased DA responsiveness to the atypical antipsychotic drugs clozapine and olanzapine may explain, at least in part, clozapine- and olanzapine-induced reversal of some of the major behavioral components of the social isolation syndrome, namely hyperactivity and attention deficit.
Amino Acids↗
Long-term evaluation of isolation-rearing induced prepulse inhibition deficits in rats.
RATIONALE: Rats reared in social isolation from weaning show prepulse inhibition (PPI) deficits which are thought to model the sensorimotor gating deficits seen in schizophrenia and other psychiatric disorders. However, recent studies have questioned the robustness of this paradigm. OBJECTIVE: The existence of a substantial dataset generated over 4 years in our laboratory has allowed the investigation of the robustness and reliability of the procedure under a variety of environmental conditions. The effects of atypical antipsychotics (clozapine, olanzapine and risperidone) under different experimental conditions are also reported. METHOD: At weaning, Hooded Lister pups were singly (isolates) or group (n=5) housed (grouped). Eight weeks later, the startle and PPI response of isolates and grouped rats was investigated using conditions of fixed inter-stimulus interval (ISI) (pulse=110 dB/50 ms; prepulse=80 dB/30 ms; ISI=100 ms) or variable
Acoustic Stimulation↗
Iatrogenic vascular injuries from percutaneous vascular suturing devices.
OBJECTIVE: The purpose of this study was to examine the patterns of injury and the strategies of surgical repair of iatrogenic vascular injuries from a percutaneous vascular suturing device after arterial cannulation. METHODS: We retrospectively reviewed the clinical experience from an academic vascular surgical practice over a 2-year period. The subjects were patients undergoing vascular repair of iatrogenic vascular injury after deployment of a percutaneous vascular suturing device. Interventions were direct repair of arterial injury (with or without device extraction) or arterial thrombectomy and repair. The main outcome variables included patterns of arterial injury, magnitude of arterial repair, limb salvage, hospital stay, and perioperative mortality and morbidity rates. RESULTS: From August 1998 through August 2000, eight patients (4 men, 4 women; median age, 55 years; range, 44-80 years) required vascular operations for complications of percutaneous suturing devices after diagnostic (2) or therapeutic (6) arteriograms through a transfemoral approach. Complications included four pseudoaneurysms (1 infected) due to arterial tear from suture pull through, two entrapped closure devices due to device malfunction, and two arterial thromboses due to narrowing/severe intimal dissection. All patients required operative intervention. Direct suture repair with or without device removal was performed in five patients, arterial debridement with vein patch angioplasty in one patient, and arterial thrombectomy and vein patch angioplasty in two patients. There were no perioperative deaths. The median hospital stay was 5 days (range, 2-33). Limbs were salvaged in all patients with a mean follow-up of 4.8 months (range, 1-13). CONCLUSIONS: Although abbreviated postangiography recovery periods and early ambulation have motivated the widespread use of percutaneous suturing devices, the infrequent occurrence of vascular injuries produced by these devices can be significantly more challenging than simple acute pseudoaneurysms or hemorrhage. In addition, thrombotic complications have a small but finite risk of limb loss.
Adult↗
Pulmonary hypertension secondary to left-sided heart disease: a cause for ventilation-perfusion mismatch mimicking pulmonary embolism.
Ventilation-perfusion (V/Q) scans are commonly performed in patients with suspected pulmonary thromboembolism (PE). V/Q mismatch is typically attributed to PE. We describe a case in which a V/Q scan performed on a patient with advanced hypertrophic obstructive cardiomyopathy showed large areas of V/Q mismatch not due to PE. The mismatch was due to pulmonary hypertension secondary to left-sided heart disease. The pathophysiology is briefly reviewed.
Adult↗
Structural studies of the high mobility group globular domain and basic tail of HMG-D bound to disulfide cross-linked DNA.
HMG-D is an abundant high mobility group chromosomal protein present during early embryogenesis in Drosophila melanogaster. It is a non-sequence-specific member of a protein family that uses the HMG domain for binding to DNA in the minor groove. The highly charged C-terminal tail of HMG-D contains AK motifs that contribute to high-affinity non-sequence-specific DNA binding. To understand the interactions of the HMG domain and C-terminal tail of HMG-D with DNA in solution, a complex between a high-affinity truncated form of the protein and a disulfide cross-linked DNA fragment was studied using heteronuclear NMR techniques. Despite its relatively high affinity for the single "prebent" site on the DNA, K(d) = 1.4 nM, HMG-D forms a non-sequence-specific complex with the DNA as indicated by exchange broadening of the protein resonances at the DNA interface in solution. The secondary structural elements of the protein are preserved when the protein is complexed with the DNA, and the DNA-binding interface maps to the regions of the protein where the largest chemical shift differences occur. The C-terminal tail of HMG-D confers high-affinity DNA binding, has an undefined structure, and appears to make direct contacts in the major groove of DNA via residues that are potentially regulated by phosphorylation. We conclude that while the HMG domain of HMG-D recognizes DNA with a mode of binding similar to that used by the sequence-specific HMG domain transcription factors, there are noteworthy differences in the structure and interactions of the C-terminal end of the DNA-binding domain and the C-terminal tail.
Amino Acid Sequence↗
Evaluation of the spontaneously hypertensive rat as a model of attention deficit hyperactivity disorder: acquisition and performance of the DRL-60s test.
CD. However, the pattern of responding at DRL-60s suggested poor schedule control for the WKY rats. Therefore, the performance of SHR in the DRL test does not appear to represent a valid model of ADHD. Further, our findings with the WKY rat suggest that this strain is a poor behavioural control for the SHR.
Animals↗
Characterization of the binding of [(125)I]-human prolactin releasing peptide (PrRP) to GPR10, a novel G protein coupled receptor.
GPR10 is a novel G-protein coupled receptor that is the human orthologue of rat Unknown Hypothalamic Receptor-1 (UHR-1). Human prolactin-releasing peptide (PrRP) has been identified as an endogenous ligand for GPR10, and occurs as 31 and 20 amino acid forms. The present study characterizes the binding of [(125)I]-PrRP-20 to HEK293 cells stably expressing GPR10 receptors. Specific binding of [(125)I]-PrRP-20 was saturable, and analysis suggested evidence of both high and low affinity sites, with K:(D:) values of 0.026+/-0.006 and 0.57+/-0.14 nM respectively, and B(max) values of 3010+/-400 and 8570+/-2240 fmol mg protein(-1) respectively. Kinetic studies were unable to distinguish two sites, but single site analysis of association and dissociation data produced a K:(D:) of 0.012 nM. Competition studies revealed that human and rat PrRP-20 and PrRP-31 all display high affinity for GPR10. A range of other drugs which are known ligands at receptors which share limited homology with GPR10 were also tested. None of the drugs tested, including the RF-amide neuropeptide FF, demonstrated any affinity for GPR10. Human PrRP-20 failed to alter basal or forskolin-stimulated levels of intracellular cyclic AMP in HEK293-GPR10 cells, suggesting that GPR10 does not couple via either G(s) or G(i). Functional studies using measurements of intracellular calcium confirmed that human and rat PrRP-20 and PrRP-31 are all potent, full agonists at the GPR10 receptor. The response was blocked both by thapsigargin, indicating mobilization of intracellular Ca(2+) stores. These studies indicate that [(125)I]-PrRP-20 is a specific, high affinity radioligand for GPR10. The availability of this radioligand binding assay will be a valuable tool for the investigation of the key features involved in PrRP binding and studies on the localization and function of GPR10.
Amino Acid Sequence↗
Radiologist workforce issues.
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Australia↗
1998 Australian radiology workforce report.
The 1998 Australian Radiologist Workforce Survey findings form the basis of the present report, prepared by the Workforce Committee of the Royal Australian and New Zealand College of Radiologists (RANZCR). A more detailed report can be accessed through the RANZCR web site (http:@www.ranzcr.edu.au). This follows similar previously published reports by the Workforce Committee in the 1990s. The issues of radiologist workforce supply and demand are examined, and benchmarks again reviewed. One of the conclusions of the present report is a possible upcoming shortage of radiologists in Australia, due to several factors, including a possible increase in the attrition rate of an ageing radiologist population, increasing female participation in the workforce, and increasing medical imaging utilization rates. In comparison with other Organization for Economic Cooperation and Development (OECD) nations, Australia had a mid-range supply of radiologists, and also medical imaging utilization, although there has been an increase in the latter. Australian radiologist annual workloads appear to be increasing.
Adult↗
Subsequent investigation and management of patients with intermediate-category and -probability ventilation-perfusion scintigraphy.
The authors wished to determine the proportion of patients with intermediate-category and intermediate-probability ventilation-perfusion scintigraphy (IVQS) who proceed to further imaging for investigation of thromboembolism, to identify the defining clinical parameters and to determine the proportion of patients who have a definite imaging diagnosis of thromboembolism prior to discharge from hospital on anticoagulation therapy. One hundred and twelve VQS studies performed at the Flinders Medical Centre over a 9-month period were reported as having intermediate category and probability for pulmonary embolism. Medical case notes were available for review in 99 of these patients and from these the pretest clinical probability, subsequent patient progress and treatment were recorded. Eight cases were excluded because they were already receiving anticoagulation therapy. In the remaining 91 patients the pretest clinical probability was considered to be low in 25; intermediate in 30; and high in 36 cases. In total, 51.6% (n = 47) of these patients (8% (n = 2) with low, 66% (n = 20) with intermediate, and 69.4% (n = 25) with high pretest probability) proceeded to CT pulmonary angiography (CTPA) and/or lower limb duplex Doppler ultrasound (DUS) evaluation. Of the patients with IVQS results, 30.7% (n = 28) were evaluated with CTPA. No patient with a low, all patients with a high and 46% of patients with an intermediate pretest probability initially received anticoagulation therapy. This was discontinued in three patients with high and in 12 patients with intermediate clinical probability prior to discharge from hospital. Overall, 40% of patients discharged on anticoagulation therapy (including 39% of those with a high pretest probability) had a positive imaging diagnosis of thromboembolism The results suggest that, although the majority of patients with intermediate-to-high pretest probability and IVQS proceed to further imaging investigation, CTPA is relatively underused in this group. Most patients with a high pretest clinical probability receive anticoagulation therapy irrespective of imaging findings, and less than half of all patients discharged from hospital on anticoagulation therapy have a positive imaging diagnosis of thromboembolism.
Angiography↗
Cellular uptake and spread of the cell-permeable peptide penetratin in adult rat brain.
Investigation of normal and pathological diseases of the central nervous system (CNS) has been hampered by the inability to effectively manipulate protein function in vivo. In order to address this important topic, we have evaluated the ability of penetratin, a novel cell-permeable peptide consisting of a 16-amino acid sequence derived from a Drosophila homeodomain protein, to act as a carrier system to introduce a cargo into brain cells. Fluorescently tagged penetratin was injected directly into rat brain, either into the striatum or the lateral ventricles, and rats were perfusion-fixed 24 h later in order to assess the brain response to the peptide. Immunohistochemistry following intrastriatal injection showed that injection of 10 microg penetratin caused neurotoxic cell death and triggered recruitment of inflammatory cells in a dose-dependent fashion. Doses of 1 microg or less resulted in reduced toxicity and recruitment of inflammatory cells, but interestingly, there was some spread of the penetratin. Injections of an inactive peptide sequence, derived from the same homeodomain, caused little toxicity but could still, however, trigger an inflammatory response. Intraventricular injections showed extensive inflammatory cell recruitment but minimal spread of either peptide. These results suggest that a dose of 1 microg of penetratin peptide is suitable for directing agents to small, discrete areas of the brain and as such is an interesting new system for analysing CNS function.
Age Factors↗
Skin perfusion pressure of the foot is a good substitute for toe pressure in the assessment of limb ischemia.
PURPOSE: Noninvasive measurements of limb systolic pressures are used routinely in the assessment of the severity of peripheral arterial disease, including the evaluation for critical limb ischemia. However, ankle pressures cannot be measured reliably in patients with medial calcification, which is especially common among patients with diabetes. Skin lesions on the toes or previous digital amputations may preclude the measurement of toe pressures. Measurements of skin perfusion pressure (SPP) are not subject to such limitations and were shown to be useful in the assessment of the severity of peripheral arterial disease. Because toe pressure is often used in the evaluation of severity of arterial disease and in the assessment for critical ischemia, we undertook to study whether there is a sufficient correlation between toe pressure and foot SPP that would allow the use of SPP measurements when toe pressures cannot be measured. METHODS: Measurements were carried out in 85 limbs of 71 patients referred to the vascular laboratory for evaluation for peripheral arterial disease. Diabetes mellitus was present in 43 patients. Each patient had foot SPP and toe pressure measurements. Toe pressures measured with photoplethysmography were correlated with foot SPP measured with laser Doppler scanning. RESULTS: There was a strong linear correlation between SPP and toe pressure (r = 0.87; P <.01). Also, significant correlation was found in both the patients with diabetes and the patients without diabetes (r = 0.85 and 0.93, respectively; P <.01 in both cases). CONCLUSIONS: We concluded that SPP measured in the foot correlates well with toe pressure and can be substituted for toe pressure measurement in patients in whom toe pressures cannot be measured.
Adult↗
Practical and theoretical issues in gene-targeting studies and their application to behaviour.
Some of the major practical and theoretical issues that are associated with gene-targeting studies in mice are discussed. The availability of sufficient space to house the extensive breeding colonies associated with studies in gene-manipulated mice is an important logistical consideration that requires consideration at an early stage. A practical example is discussed which illustrates some of these issues. Problems associated with disease control and methods of maintaining the health status of valuable colonies are also outlined. Differences in the behavioural phenotype of inbred mouse strains pose important issues for study design and selection of host mouse lines. The results from studies exploring variations in the behavioural phenotype of six common inbred strains are briefly outlined. The impact of phenotypic variation on behavioural studies is considered and the implications for experimental design are discussed.
Animals↗
Pharmacological actions of a novel, high-affinity, and selective human dopamine D(3) receptor antagonist, SB-277011-A.
SB-277011-A (trans-N-[4-[2-(6-cyano-1,2,3, 4-tetrahydroisoquinolin-2-yl)ethyl]cyclohexyl]-4-quinolininecarboxamide), is a brain-penetrant, high-affinity, and selective dopamine D(3) receptor antagonist. Radioligand-binding experiments in Chinese hamster ovary (CHO) cells transfected with human dopamine D(3) or D(2 long) (hD(3), hD(2)) receptors showed SB-277011-A to have high affinity for the hD(3) receptor (pK(i) = 7.95) with 100-fold selectivity over the hD(2) receptor and over 66 other receptors, enzymes, and ion channels. Similar radioligand-binding data for SB-277011-A were obtained from CHO cells transfected with rat dopamine D(3) or D(2). In the microphysiometer functional assay, SB-277011-A antagonized quinpirole-induced increases in acidification in CHO cells overexpressing the hD(3) receptor (pK(b) = 8.3) and was 80-fold selective over hD(2) receptors. Central nervous system penetration studies showed that SB-277011-A readily entered the brain. In in vivo microdialysis studies, SB-277011-A (2. 8 mg/kg p.o.) reversed the quinelorane-induced reduction of dopamine efflux in the nucleus accumbens but not striatum, a regional selectivity consistent with the distribution of the dopamine D(3) receptor in rat brain. SB-277011-A (2-42.3 mg/kg p.o.) did not affect spontaneous locomotion, or stimulant-induced hyperlocomotion. SB-277011-A (4.1-42.2 mg/kg p.o.) did not reverse prepulse inhibition deficits in apomorphine- or quinpirole-treated rats, but did significantly reverse the prepulse inhibition deficit in isolation-reared rats at a dose of 3 mg/kg p.o. SB-277011-A (2.5-78. 8 mg/kg p.o.) was noncataleptogenic and did not raise plasma prolactin levels. Thus, dopamine D(3) receptor blockade produces few of the behavioral effects characteristic of nonselective dopamine receptor antagonists. The effect of SB-277011-A on isolation-induced prepulse inhibition deficit suggests that blockade of dopamine D(3) receptors may benefit the treatment of schizophrenia.
Animals↗