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Biomedical subjects

D Smart

Publications and source records attributed to D Smart.

At least 37 records · Page 2Linked to original sources
PubMed2001

SB-334867-A: the first selective orexin-1 receptor antagonist.

The pharmacology of various peptide and non-peptide ligands was studied in Chinese hamster ovary (CHO) cells stably expressing human orexin-1 (OX(1)) or orexin-2 (OX(2)) receptors by measuring intracellular calcium ([Ca(2+)](i)) using Fluo-3AM. Orexin-A and orexin-B increased [Ca(2+)](i) in CHO-OX(1) (pEC(50)=8.38+/-0.04 and 7.26+/-0.05 respectively, n=12) and CHO-OX(2) (pEC(50)=8.20+/-0.03 and 8.26+/-0.04 respectively, n=8) cells. However, neuropeptide Y and secretin (10 pM - 10 microM) displayed neither agonist nor antagonist properties in either cell-line. SB-334867-A (1-(2-Methyylbenzoxanzol-6-yl)-3-[1,5]naphthyridin-4-yl-urea hydrochloride) inhibited the orexin-A (10 nM) and orexin-B (100 nM)-induced calcium responses (pK(B)=7.27+/-0.04 and 7.23+/-0.03 respectively, n=8), but had no effect on the UTP (3 microM)-induced calcium response in CHO-OX(1) cells. SB-334867-A (10 microM) also inhibited OX(2) mediated calcium responses (32.7+/-1.9% versus orexin-A). SB-334867-A was devoid of agonist properties in either cell-line. In conclusion, SB-334867-A is a non-peptide OX(1) selective receptor antagonist.

Animals↗
PubMed2001

Longitudinal predictors of behavioural adjustment in pre-adolescent children.

OBJECTIVES: Longitudinal data from infancy onwards, from the Australian Temperament Project, a prospective study of the temperament and development of a large and representative sample of Victorian children, were examined to identify predictors of psychological disorder at 11-12 years of age. METHOD: Those children scoring in the at-risk range for psychological disorder according to parents, teachers and self-reports using the Child Behaviour Questionnaire were selected at 11-12 years of age for in-depth assessment and comparison with a group of children with no history of adjustment problems. Analyses of group differences using longitudinal data gathered from infancy to 12 years focused on parent and teacher reports on child temperament and behaviour, and various facets of home and school adjustment. RESULTS: The strongest predictors of adjustment at 12 years were previous behaviour problems, along with some specific temperament factors involving self-regulation capacities and mother's overall rating of child difficulty. Results based on parallel teacher data including peer adjustment, and social and academic competence measures were consistent with parent data. CONCLUSIONS: Our research confirms the persistence of early appearing behaviour problems in a community sample and the longitudinal influence of temperament factors in childhood. The study supports the need for a focus on early intervention and prevention strategies in the child mental health field.

Adolescent↗
PubMed2000

Characterisation of an endogenous bombesin receptor in CHO/DG44 cells.

Bombesin and its receptors have been shown to have a role regulating circadian rhythms in the hamster suprachiasmatic and dorsal raphe nuclei and have been implicated in the regulation of sleep. We have identified and characterised a bombesin receptor endogenously expressed in a Chinese hamster ovary cell line (CHO/DG44). Using a range of bombesin-like peptides, we demonstrate that this receptor displays bombesin BB2 receptor-like pharmacology. We also show that this receptor signals through inositol-[1,4,5]-trisphosphate and protein kinase C and thus provides a useful model system to aid in the interpretation of hamster suprachiasmatic nucleus studies of mammalian circadian rhythm.

Animals↗
PubMed2000

A longitudinal study of the role of childhood temperament in the later development of eating concerns.

OBJECTIVE: This study examined the relationship between childhood temperament and the later development of eating and body concerns in early adolescent children. METHOD: The Australian Temperament Project has followed a cohort of children from birth, assessing temperament factors such as Negative Emotionality, Persistence, Approach/Withdrawal, and Activity. Using a longitudinal design, the study reports on the relationship between temperament measured from infancy onward and eating and body concerns at 12-13 years of age. Participants (597 girls, 631 boys) completed the Eating Disorders Inventory subscales Drive For Thinness, Body Dissatisfaction, and Bulimia, and an estimate of their current size. Parents described their child's temperament and body size. RESULTS: High Negative Emotionality and low Persistence were the factors most associated with risk status over time, particularly in girls. DISCUSSION: While it is unlikely that temperamental characteristics per se lead to disordered eating, it is argued that in combination with other risk factors, certain temperamental characteristics may increase vulnerability.

Adolescent↗
PubMed2000

Removal of the subchondral plate in acetabular preparation.

Retention of the subchondral plate during acetabular preparation in total hip replacement is believed to be an important part of modern cementing techniques. We have constructed a two-dimensional finite element analysis to assess the effect of retention and removal of this relatively stiff structure. The finite element analysis demonstrates increased stiffness and stress concentrations at the bone-cement interface that may have an adverse effect. Although further study is required, it may be that subchondral bone retention is not advantageous.

Acetabulum↗
PubMed2000

The endogenous lipid anandamide is a full agonist at the human vanilloid receptor (hVR1).

The endogenous cannabinoid anandamide was identified as an agonist for the recombinant human VR1 (hVR1) by screening a large array of bioactive substances using a FLIPR-based calcium assay. Further electrophysiological studies showed that anandamide (10 or 100 microM) and capsaicin (1 microM) produced similar inward currents in hVR1 transfected, but not in parental, HEK293 cells. These currents were abolished by capsazepine (1 microM). In the FLIPR anandamide and capsaicin were full agonists at hVR1, with pEC(50) values of 5. 94+/-0.06 (n=5) and 7.13+/-0.11 (n=8) respectively. The response to anandamide was inhibited by capsazepine (pK(B) of 7.40+/-0.02, n=6), but not by the cannabinoid receptor antagonists AM630 or AM281. Furthermore, pretreatment with capsaicin desensitized the anandamide-induced calcium response and vice versa. In conclusion, this study has demonstrated for the first time that anandamide acts as a full agonist at the human VR1.

Amides↗
PubMed2000

The hypocretins are weak agonists at recombinant human orexin-1 and orexin-2 receptors.

The pharmacology of the orexin-like peptides, hypocretin-1 and hypocretin-2, was studied in Chinese hamster ovary (CHO) cells stably expressing orexin-1 (OX(1)) or orexin-2 (OX(2)) receptors by measuring intracellular calcium ([Ca(2+)](i)) using Fluo-3AM. Orexin-A and orexin-B increased [Ca(2+)](i) in CHO-OX(1) (pEC(50)=7. 99+/-0.05 and 7.00+/-0.10 respectively, n=8) and CHO-OX(2) (pEC(50)=8.30+/-0.05 and 8.21+/-0.07 respectively, n=5). However, hypocretin-1 and hypocretin-2 were markedly less potent, with pEC(50) values of 5.31+/-0.04 and 5.41+/-0.04 respectively in CHO-OX(2) cells (n=5). In CHO-OX(1) cells 10 microM hypocretin-1 only elicited a 37.5+/-3.4% response whilst 10 microM hypocretin-2 elicited a 18.0+/-2.1% response (n=8). Desensitisation of OX(1) or OX(2) with orexin-A (100 nM) abolished the response to orexin-A (10 nM) and the hypocretins (10 microM), but not to UTP (3 microM). In conclusion, the hypocretins are only weak agonists at the orexin receptors.

Aniline Compounds↗
PubMed2000

Characterization using FLIPR of rat vanilloid receptor (rVR1) pharmacology.

The vanilloid receptor (VR1) is a ligand-gated ion channel, which plays an important role in nociceptive processing. Therefore, a pharmacological characterization of the recently cloned rat VR1 (rVR1) was undertaken. HEK293 cells stable expressing rVR1 (rVR1-HEK293) were loaded with Fluo-3AM and then incubated at 25 degrees C for 30 min with or without various antagonists or signal transduction modifying agents. Then intracellular calcium concentrations ([Ca(2+)](i)) were monitored using FLIPR, before and after the addition of various agonists. The rank order of potency of agonists (resiniferatoxin (RTX)>capsaicin>olvanil>PPAHV) was as expected, and all were full agonists. The potencies of capsaicin and olvanil, but not RTX or PPAHV, were enhanced at pH 6.4 (pEC(50) values of 7.47+/-0.06, 7.16+/-0.06, 8.19+/-0.06 and 6.02+/-0.03 respectively at pH 7.4 vs 7.71+/-0.05, 7.58+/-0.14, 8.10+/-0.05 and 6.04+/-0.08 at pH 6.4). Capsazepine, isovelleral and ruthenium red all inhibited the capsaicin (100 nM)-induced Ca(2+) response in rVR1-HEK293 cells, with pK(B) values of 7.52+/-0.08, 6.92+/-0.11 and 8.09+/-0.12 respectively (n=6 each). The response to RTX and olvanil were also inhibited by these compounds. None displayed any agonist-like activity. The removal of extracellular Ca(2+) abolished, whilst inhibition of protein kinase C with chelerythrine chloride (10 microM) partially (approximately 20%) inhibited, the capsaicin (10 microM)-induced Ca(2+) response. However, tetrodotoxin (3 microM), nimodipine (10 microM), omega-GVIA conotoxin (1 microM), thapsigargin (1 microM), U73122 (3 microM) or H-89 (3 microM) had no effect on the capsaicin (100 nM)-induced response. In conclusion, the recombinant rVR1 stably expressed in HEK293 cells acts as a ligand-gated Ca(2+) channel with the appropriate agonist and antagonist pharmacology, and therefore is a suitable model for studying the effects of drugs at this receptor.

Animals↗
PubMed2000

Association of a functional polymorphism of the serotonin transporter gene with anxiety-related temperament and behavior problems in children: a longitudinal study from infancy to the mid-teens.

A polymorphism in the promoter region of the serotonin transporter gene (5-HTTLPR) has been associated with anxiety-related personality traits in adults. Initial studies showed that the short allele was associated with higher neuroticism, anxiety and harm avoidance. However, most attempts to replicate these findings have been negative. Because the association of candidate polymorphisms with behavioral traits may vary with stage of development, we investigated the association using participants in a longitudinal study of childhood temperament. DNA was available for 660 children who had been assessed for temperament from 4-8 months to 15-16 years, and for behaviour problems from 3-4 years to 15-16 years. No significant associations were found at most ages. However, at ages 13-14 years and 15-16 years, the long/long genotype was associated with higher anxiety. These findings do not support an association of the short allele with anxiety-related traits in early life.

Adolescent↗
PubMed2000

Does shy-inhibited temperament in childhood lead to anxiety problems in adolescence?

OBJECTIVE: To assess the relationships between shy-inhibited temperament in childhood and anxiety problems in early adolescence using a prospective, longitudinal data set from a large community sample. METHOD: Relationships between shyness ratings on age-appropriate temperament scales and anxiety problems were analyzed, looking both forward and backward in time from infancy to adolescence. RESULTS: Forty-two percent of children rated as shy on 6 or more occasions over 8 surveys in childhood had anxiety problems in adolescence, compared with 11% who were never shy. Persistence of shyness and its presence in middle childhood increased risk for anxiety. A highly reactive temperament added to shyness did not increase the risk for anxiety. Few children with an anxiety diagnosis in early adolescence had a history of shyness. CONCLUSIONS: Prediction from childhood shyness to adolescent anxiety disorder is modest but clinically meaningful in a community sample. However, most shy children did not develop an anxiety disorder and most adolescents with anxiety disorders had not been especially shy.

Adolescent↗
PubMed2000

Cytosensor techniques for examining signal transduction of neurohormones.

This review describes the principles of microphysiometry and how they can be applied, using the Cytosensor, to the investigation of the signal transduction mechanisms activated by both G-protein and non-G-protein coupled hormone and neuropeptide receptors. The use of the Cytosensor to study desensitisation and cross-talk is also discussed, as are the benefits and limitations of this technique.

Animals↗
PubMed1999

Characterisation using microphysiometry of CRF receptor pharmacology.

We have assessed the utility of the Cytosensor microphysiometer for studying the pharmacology of recombinant CRF receptors. Chinese hamster ovary cells stably expressing the human CRF1 or CRF2 receptor were perfused in the Cytosensor with bicarbonate-free Hams F12 (pH 7.4) containing 0.2% bovine serum albumin. The rank order of potencies of agonist peptides were CRF = sauvagine = urocortin = urotensin at CRF1 (pEC50 values 11.16 +/- 0.17, 11.37 +/- 0.14, 11.43 +/- 0.09 and 11.46 +/- 0.13; n = 4), and urocortin = sauvagine > urotensin > CRF at CRF2 (pEC50 values 10.88 +/- 0.12, 10.44 +/- 0.05, 9.36 +/- 0.12 and 8.53 +/- 0.07; n = 7-9). alpha-Helical CRF (9-41) was a competitive antagonist at the CRF2 receptor (pK(B) = 6.99 +/- 0.08, n = 4), but was a partial agonist at the CRF1 receptor (pEC50 = 6.85 +/- 0.08, Emax = 33%, n = 3). CP 154,526 was a competitive antagonist at the CRF1 receptor (pK(B) = 8.17 +/- 0.05, n = 6), but was inactive at the CRF2 receptor. These data are consistent with established CRF receptor pharmacology and show that the Cytosensor is a viable method for assessing the functional activity of CRF-receptor agonists and antagonists.

Acid-Base Equilibrium↗
PubMed1999

Rainbow trout (Oncorhynchus mykiss) urotensin-I: structural differences between urotensins-I and urocortins.

In bony fishes, both corticotropin-releasing factor (CRF) and urotensin-I play a role in the regulation of interrenal glucocorticoid release. The rainbow trout, Oncorhynchus mykiss, is a useful model for understanding the mechanisms of stress and the hypothalamo-pituitary-interrenal axis because of its phylogenetic position at the base of the euteleostei and its popularity as a food fish. Urotensin-I may act as a glucocorticoid releaser in a mechanism phylogenetically older than that of CRF. The structural and functional relationships of trout urotensin-I have been investigated. The transcript was cloned from a trout brain hypothalamic cDNA library. A single positive clone was isolated and sequenced. It possesses 3218 bases and has the longest 3' untranslated region of all urotensins-I and CRF transcripts found to date. In comparison to the other fish orthologues, it has the closest sequence identity to the mammalian urocortins. The transcript appears to be differentially processed in brain and urophysis as determined by Northern blot analysis and the presence of polyadenylation signals in the 3' untranslated region. Synthetic trout urotensin-I activated both human CRF-R1 and -R2 receptor-transfected CHO cells with a potency similar to that of white sucker (Catostomus commersoni) urotensin-I. Both fish neuropeptides possessed an order of magnitude less potency than human urocortin in CRF-R2 transfected cells.

Amino Acid Sequence↗
PubMed1999

Abductor weakness and stresses around acetabular components of total hip arthroplasty: a finite element analysis.

Abductor weakness, and the resulting Trendelenburg gait, after total hip arthroplasty is believed to be associated with a poor long-term outcome. We have constructed a two-dimensional finite element analysis using load cases to mimic this abductor weakness. The finite element analysis demonstrates slightly increased stresses, particularly at the bone-cement interface in the DeLee-Charnley zone I, which does not seem sufficient to explain the adverse effect of abductor weakness.

Acetabulum↗
PubMed1999

Cloning and characterization of corticotropin-releasing factor and urocortin in Syrian hamster (Mesocricetus auratus).

Corticotropin-releasing factor and urocortin belong to a superfamily of neuropeptides that includes the urotensins-I in fishes and the insect diuretic peptides. Sequence analysis suggests that urocortin is the mammalian ortholog of urotensin-I, although the physiological role for this peptide in mammals is not known. Within the Rodentia, hamsters belong to a phylogenetically older lineage than that of mice and rats and possess significant differences in hypothalamic organization. We have, therefore, cloned the coding region of the Syrian hamster (Mesocricetus auratus) corticotropin-releasing factor and urocortin mature peptide by polymerase chain reaction. Hamster urocortin was prepared by solid-phase synthesis, and its pharmacological actions on human corticotropin-releasing factor R1 and R2 receptors were investigated. The deduced hamster corticotropin-releasing factor amino acid sequence and cleavage site is identical to that in rat, whereas the urocortin sequence is unique among the urocortin/urotensin-I/sauvagine family in possessing asparagine and alanine in positions 38 and 39, respectively. The hamster urocortin carboxy terminus sequence bears greater structural similarity to the insect diuretic peptide family, suggesting either retrogressive mutational changes within the mature peptide or convergent sequence evolution. Despite these changes, human and hamster urocortin are generally equipotent at cAMP activation, neuronal acidification rate, and R1/R2 receptor affinities.

Amino Acid Sequence↗
PubMed1999

Comparison of the effects of [Phe1psi(CH2-NH)Gly2]nociceptin(1-13)NH2 in rat brain, rat vas deferens and CHO cells expressing recombinant human nociceptin receptors.

Nociceptin(NC) is the endogenous ligand for the opioid receptor like-1 receptor (NC-receptor). [Phe1(psi)(CH2-NH)Gly2]Nociceptin(1-13)NH2 ([F/G]NC(1-13)NH2) has been reported to antagonize NC actions in peripheral guinea-pig and mouse tissues. In this study, we investigated the effects of a range of NC C-terminal truncated fragments and [F/G]NC(1-13)NH2 on NC receptor binding, glutamate release from rat cerebrocortical slices (rCX), inhibition of cyclic AMP accumulation in CHO cells expressing the NC receptor (CHO(NCR)) and electrically evoked contractions of the rat vas deferens (rVD). In radioligand binding assays, a range of ligands inhibited [125I]-Tyr14-NC binding in membranes from rCX and CHO(NCR) cells. As the peptide was truncated there was a general decline in pKi. [F/G]NC(1-13)NH2 was as potent as NC(1-13)NH2. The order of potency for NC fragments to inhibit cyclic AMP accumulation in whole CHO(NCR) cells was NCNH2> or =NC=NC(1-13)NH2>NC(1-12)NH2> >NC(1-11)NH2. [F/G]NC(1-13)NH2 was a full agonist with a pEC50 value of 8.65. NCNH2 and [F/G]NC(1-13)NH2 both inhibited K+ evoked glutamate release from rCX with pEC50 and maximum inhibition of 8.16, 48.5+/-4.9% and 7.39, 58.9+/-6.8% respectively. In rVD NC inhibited electrically evoked contractions with a pEC50 of 6.63. Although [F/G]NC(1-13)NH2, displayed a small (instrinsic activity alpha = 0.19) but consistent residual agonist activity, it acted as a competitive antagonist (pA2 6.76) in the rVD. The differences between [F/G]NC(1-13)NH2 action on central and peripheral NC signalling could be explained if [F/G]NC(1-13)NH2 was a partial agonist with high strength of coupling in the CNS and low in the periphery. An alternative explanation could be the existence of central and peripheral receptor isoforms.

Animals↗
PubMed1999

Characterization of recombinant human orexin receptor pharmacology in a Chinese hamster ovary cell-line using FLIPR.

The cellular mechanisms underlying the physiological effects of the orexins are poorly understood. Therefore, the pharmacology of the recombinant human orexin receptors was studied using FLIPR. Intracellular calcium ([Ca2+]i) was monitored in Chinese hamster ovary (CHO) cells stably expressing orexin-1 (OX1) or orexin-2 (OX2) receptors using Fluo-3AM. Orexin-A and orexin-B increased [Ca2+]i in a concentration dependent manner in CHO-OX1 (pEC50=8.03+/-0.08 and 7. 30+/-0.08 respectively, n=5) and CHO-OX2 (pEC50=8.18+/-0.10 and 8. 43+/-0.09 respectively, n=5) cells. This response was typified as a rapid peak in [Ca2+]i (maximal at 6 - 8 s), followed by a gradually declining secondary phase. Thapsigargin (3 microM) or U73122 (3 microM) abolished the response. In calcium-free conditions the peak response was unaffected but the secondary phase was shortened, returning to basal values within 90 s. Calcium (1.5 mM) replacement restored the secondary phase. In conclusion, orexins cause a phospholipase C-mediated release of calcium from intracellular stores, with subsequent calcium influx.

Aniline Compounds↗