Swimming behaviour and post-swimming activity in Vitamin D receptor knockout mice

Animal experiments have shown that Vitamin D plays a role in both brain development and adult brain function. The adult Vitamin D receptor null mutant mouse (VDR -/-) is reported to be less active and more anxious than wild-type litter mate controls and to have poor swimming ability. However, an anxious behavioural phenotype is inferred from differences in locomotor behaviour. This is a general problem in behavioural phenotyping where a neurological phenotype is inferred from changes in locomotion which will be affected by non-neurological factors, such as muscle fatigue. In this study of VDR -/-, we conducted a detailed examination of one form of motor behaviour, swimming, compared to wildtype littermate controls. Swimming was assessed using a forced swim test, a laneway swimming test and a watermaze test using a visible platform. Post-swimming activity was assessed by comparing grooming and rearing behaviour before, and 5 min after, the forced swimming test. We replicated previous findings in which VDR -/- mice demonstrate more sinking episodes than wildtype controls in the forced swim test but they were similar to controls in the time taken to swim a 1 m laneway, and in the time taken to reach a visible platform in the watermaze. Thus, the VDR -/- mice were able to swim but were not able to float. Grooming and rearing behaviour of the VDR -/- mice was similar to wildtype controls before the forced swim but the VDR -/- were much less active after the swim compared with wildtype mice which displayed high levels of grooming and rearing. We conclude that VDR -/- mice have muscular and motor impairments that do not affect their ability to swim but significantly alters the ability to float as well as their post-swimming activity. Differences in muscle strength may confound tests of activity that are used to infer an anxious phenotype. (c) 2005 Elsevier Inc. All rights reserved.

The mouse sulfate anion transporter gene Sat1 (Slc26a1): Cloning, tissue distribution, gene structure, functional characterization, and transcriptional regulation by thyroid hormone

Sulfate (SO42-) is required for bone/cartilage formation and cellular metabolism. sat-1 is a SO42- anion transporter expressed on basolateral membranes of renal proximal tubules, and is suggested to play an important role in maintaining SO42- homeostasis. As a first step towards studying its tissue-specific expression, hormonal regulation, and in preparation for the generation of knockout mice, we have cloned and characterized the mouse sat-1 cDNA (msat-1), gene (sat1; Slc26a1) and promoter region. msat-1 encodes a 704 amino acid protein (75.4 kDa) with 12 putative transmembrane domains that induce SO42- (also oxalate and chloride) transport in Xenopus oocytes. msat-1 mRNA was expressed in kidney, liver, cecum, calvaria, brain, heart, and skeletal muscle. Two distinct transcripts were expressed in kidney and liver due to alternative utilization of the first intron, corresponding to an internal portion of the 5'-untranslated region. The Sa1 gene (similar to6 kb) consists of 4 exons. Its promoter is similar to52% G+C rich and contains a number of well-characterized cis-acting elements, including sequences resembling hormone responsive elements T3REs and VDREs. We demonstrate that Sat1 promoter driven basal transcription in OK cells was stimulated by tri-iodothyronine. Site-directed mutagenesis identified an imperfect T3RE at -454-bp in the Sat1 promoter to be responsible for this activity. This study represents the first characterization of the structure and regulation of the Sat1 gene encoding a SO42-/chloride/oxalate anion transporter.

Physical activity promotion in primary care: Bridging the gap between research and practice

Background: Aflifle a growing literature supports the effectiveness of physical activity interventions delivered in the primary care setting, few studies have evaluated efforts to increase physician counseling on physical activity during routine practice (i.e., outside the context of controlled research). This paper reports the results of a dissemination trial of a primary care-based physical activity counseling intervention conducted within the context of a larger, multi-strategy, Australian community-based, physical activity intervention, the 10,000 Steps Rockhampton Project. Methods: All 23 general practices and 66 general practitioners (GPs, the Australian equivalent of family physicians) were invited to participate. Practice visits were made to consenting practices during which instruction in brief physical activity counseling was offered, along with physical activity promotion resources (print materials and pedometers). The evaluation, guided by the RE-AIM framework, included collection of process data, as well as pre-and post-inteivention data from a mailed GP survey, and data from the larger project's random-digit-dialed, community-based, cross-sectional telephone survey that was conducted in Rockhampton and a comparison community, Results: Ninety-one percent of practices were visited by 10,000 Steps staff and agreed to participate, with 58% of GPs present during the visits. General practitioner survey response rates were 67% (n =44/66 at baseline) and 71% (n =37/52, at 14-month follow-up). At follow-up, 62% had displayed the poster, 81% were using the brochures, and 70% had loaned pedometers to patients, although the number loaned was relatively small. No change was seen in GP self-report of the percentage of patients counseled on physical activity. However, data from the telephone surveys showed a 31% increase in the likelihood of recalling GP advice on physical activity in Rockhampton (95% confidence interval [CI]=1.11-1.54) compared to a 16% decrease (95% CI=0.68-1.04) in the comparison community. Conclusions: This dissemination study achieved high rates of GP uptake, reasonable levels of implementation, and a significant increase in the number of community residents counseled on physical activity. These results suggest that evidence-based primary care physical activity counseling protocols can be translated into routine practice, although the initial and ongoing investment of time to develop partnerships with relevant healthcare organizations, and the interest generated by the overall 10,000 Steps program should not be underestimated. ((C) 2004 American journal of Preventive Medicine.

Physical activity cost in children following an acquired brain injury - a comparative study

Background: Alterations in energy expenditure during activity post head injury has not been investigated due primarily to the difficulty of measurement. Objective: The aim of this study was to compare energy expenditure during activity and body composition of children following acquired brain injury (ABI) with data from a group of normal. controls. Design: Energy expenditure was measured using the Cosmed K4b(2) in a group of 15 children with ABI and a group of 67 normal children during rest and when walking and running. Mean number of steps taken per 3 min run was also recorded and body composition was measured. Results: The energy expended during walking was not significantly different between both groups. A significant difference was found between the two groups in the energy expended during running and also for the number of steps taken as children with ABI took significantly less steps than the normal controls during a 3 min run. Conclusions: Children with ABI exert more energy per activity than healthy controls when controlled for velocity or distance. However, they expend less energy to walk and run when they are free to choose their own desirable, comfortable pace than normal controls. (C) 2003 Elsevier Ltd. All rights reserved.

Acute cadmium chloride administration induces hepatic and renal CYP2A5 mRNA, protein and activity in the mouse: involvement of transcription factor NRF2

Modulation of the cytochrome P450 (CYP) monooxygenase system by cadmium was investigated in male, adult DBA/2J mice treated with a single dose (16 mumol/kg body weight, i.p.) of cadmium chloride (CdCl2). Total CYP content of liver and kidney microsomes decreased maximally (56% and 85%, respectively) 24 and 18 h, respectively, after CdCl2 treatment. Progressive increases of hepatic coumarin 7-hydroxylase (COH) activity; indicative of CYP2A5 activity, relative to the total CYP content were seen at 8 h (2-fold), 12 h (3-fold), 18 h (12-fold), and 24 h (15-fold). Similar changes were seen in the kidney. Liver and kidney CYP2A5 mRNA levels increased maximally 12 and 4 h after treatment and decreased to almost half 6 h later. In contrast, kidney and liver CYP2A5 protein levels increased maximally at 18 and 24 h. The CYP2A5 mRNA levels in the kidney and liver increased after Cd treatment in Nrf2 +/+ but not in Nrf2 -/- mouse. This study demonstrates that hepatic and kidney CYP2A5 is upregulated by cadmium with a somewhat faster response in the kidney than the liver. The strong upregulation of the CYP2A5 both at mRNA and enzyme activity levels, with a simultaneous decrease in the total CYP concentration suggest an unusual mode of regulation of CYP2A5 in response to cadmium exposure, amongst the CYP enzymes. The observed decrease in the mRNA but not in protein levels after maximal induction may suggest involvement of post-trancriptional mechanisms in the regulation. Upregulation of CYP2A5 by cadmium in the Nrf2 +/+ mice but not in the Nrf2 -/- mice indicates a role for this transcription factor in the regulation. (C) 2003 Elsevier Ireland Ltd. All rights reserved.

The ins and outs of transcriptional control: nucleocytoplasmic shuttling in development and disease

Recent findings relating to SOX transcription factors indicate that defects in organogenesis can be caused not only by impairment of the biochemical properties of transcription factors but also, in some cases, by deficient nuclear import. In addition, experimentally interfering with the nuclear export signals of some SOX factors has now been found to cause developmental defects. Controlling the balance of nuclear import and export might be a common means by which transcription factor activity can be regulated during development, and defects in these processes might underlie a broader spectrum of inherited developmental disorders.

Acute cadmium chloride administration induces hepatic and renal cytochrome P450 2A5 in the mouse

Modulation of the cytochrome P450 (CYP) monooxygenase system by cadmium was investigated in male, adult DBA/2J mice treated with a single dose (16 Amol/kg body weight, i.p.) of cadmium chloride (CdCl2) at various time points. The total CYP content of kidney microsomes started to decrease 4 hours earlier than in the liver (P < 0.05), with maximal decreases at 24 hours of 56% and 85% in the liver and kidney, respectively. In contrast, both hepatic and renal coumarin 7-hydroxylase (COH) activity (indicative of CYP2A5 activity) relative to total CYP content started to progressively increase at 8 hours, with renal activity 61 times higher than the hepatic activity. Maximum increases were observed, 15-fold in the liver and 64-fold in the kidney after 24 hours. Liver and kidney CYP2A5 mRNA levels increased maximally 12 and 4 hours after treatment, respectively and decreased to almost half 6 hours later. In contrast, kidney and liver CYP2A5 protein levels increased maximally at 18 and 24 hours. This study demonstrates that hepatic and renal CYP2A5 is upregulated by cadmium with a faster response in the kidney than in the liver. This observation is concordant with the fact that kidney is the target organ for cadmium toxicity. The observed increase in the mRNA but not in protein levels after maximal induction suggests involvement of post-transcriptional mechanisms in the regulation of CYP2A5 expression by cadmium.

Blunted growth hormone response to clonidine in post-traumatic stress disorder

Hyperactivity of the sympathetic and noradrenergic systems is thought to be a feature of post-traumatic stress disorder (PTSD). Assessment of noradrenergic receptor function can be undertaken by measuring the growth hormone (GH) response to the alpha(2)-agonist clonidine. The aim of this study was to examine whether subjects with combat-related PTSD (with or without co-morbid depression) have a blunted growth hormone response to clonidine, compared to a combat-exposed control group. Twenty-three Vietnam veterans suffering from PTSD alone, 27 suffering from PTSD and co-morbid depression, and 32 veteran controls with no psychiatric illness were administered 1.5 mug/kg clonidine i.v. Plasma growth hormone was measured every 20 min for 120 min. The growth hormone response to clonidine was significantly blunted in the non-depressed PTSD group compared to both the depressed PTSD group and the control group as measured by peak growth hormone, delta growth hormone and AUC growth hormone. Subjects with PTSD and no co-morbid depressive illness show a blunted growth hormone response to clonidine. This suggests that post-synaptic alpha(2)-receptors are subsensitive. This finding is consistent with other studies showing increased noradrenergic activity in PTSD. (C) 2003 Elsevier Ltd. All rights reserved.

Modulation of higher-plant NAD(H)-dependent glutamate dehydrogenase activity in transgenic tobacco via alteration of beta subunit levels

Glutamate dehydrogenase (GDH; EC 1.4.1.2-1.4.1.4) catalyses in vitro the reversible amination of 2-oxoglutarate to glutamate. In vascular plants the in vivo direction(s) of the GDH reaction and hence the physiological role(s) of this enzyme remain obscure. A phylogenetic analysis identified two clearly separated groups of higher-plant GDH genes encoding either the alpha- or beta-subunit of the GDH holoenzyme. To help clarify the physiological role(s) of GDH, tobacco (Nicotiana tabacum L.) was transformed with either an antisense or sense copy of a beta-subunit gene, and transgenic plants recovered with between 0.5- and 34-times normal leaf GDH activity. This large modulation of GDH activity (shown to be via alteration of beta-subunit levels) had little effect on leaf ammonium or the leaf free amino acid pool, except that a large increase in GDH activity was associated with a significant decrease in leaf Asp (similar to 51%, P=0.0045). Similarly, plant growth and development were not affected, suggesting that a large modulation of GDH beta-subunit titre does not affect plant viability under the ideal growing conditions employed. Reduction of GDH activity and protein levels in an antisense line was associated with a large increase in transcripts of a beta-subunit gene, suggesting that the reduction in beta-subunit levels might have been due to translational inhibition. In another experiment designed to detect post-translational up-regulation of GDH activity, GDH over-expressing plants were subjected to prolonged dark-stress. GDH activity increased, but this was found to be due more likely to resistance of the GDH protein to stress-induced proteolysis, rather than to post-translational up-regulation.

Hypoxia induces chondrocyte-specific gene expression in mesenchymal cells in association with transcriptional activation of Sox9

Endochondral bone is formed during an avascular period in an environment of low oxygen. Under these conditions, pluripotential mesenchymal stromal cells preferentially differentiate into chondrocytes and form cartilage. In this study, we investigated the hypothesis that oxygen tension modulates bone mesenchymal cell fate by altering the expression of genes that function to promote chondrogenesis. Microarray of RNA samples from ST2 cells revealed significant changes in 728 array elements (P < 0.01) in response to hypoxia. Real-time PCR on these RNA samples, and separate samples from C3H10T1/2 cells, revealed hypoxia-induced changes in the expression of additional genes known to be expressed by chondrocytes including Sox9 and its downstream targets aggrecan and Col2a. These changes were accompanied by the accumulation of mucopolysacharide as detected by alcian blue staining. To investigate the mechanisms responsible for upregulation of Sox9 by hypoxia, we determined the effect of hypoxia on HIF-1 alpha levels and Sox9 promoter activity in ST2 cells. Hypoxia increased nuclear accumulation of HIF-1 alpha and activated the Sox9 promoter. The ability of hypoxia to transactivate the Sox9 promoter was virtually abolished by deletion of HIF-1 alpha consensus sites within the proximal promoter. These findings suggest that hypoxia promotes the differentiation of mesenchymal cells along a chondrocyte pathway in part by activating Sox-9 via a HIF-1 alpha-dependent mechanism. (c) 2005 Elsevier Inc. All rights reserved.

The prion protein gene: Identifying regulatory signals using marsupial sequence

The function of the prion protein gene (PRNP) and its normal product PrPC is elusive. We used comparative genomics as a strategy to understand the normal function of PRNP. As the reliability of comparisons increases with the number of species and increased evolutionary distance, we isolated and sequenced a 66.5 kb BAC containing the PRNP gene from a distantly related mammal, the model Australian marsupial Macropus eugenii (tammar wallaby). Marsupials are separated from eutherians such as human and mouse by roughly 180 million years of independent evolution. We found that tammar PRNP, like human PRNP, has two exons. Prion proteins encoded by the tammar wallaby and a distantly related marsupial, Monodelphis domestica (Brazilian opossum) PRNP contain proximal PrP repeats with a distinct, marsupial-specific composition and a variable number. Comparisons of tammar wallaby PRNP with PRNPs from human, mouse, bovine and ovine allowed us to identify non-coding gene regions conserved across the marsupial-eutherian evolutionary distance, which are candidates for regulatory regions. In the PRNP 3' UTR we found a conserved signal for nuclear-specific polyadenylation and the putative cytoplasmic polyadenylation element (CPE), indicating that post-transcriptional control of PRNP mRNA activity is important. Phylogenetic footprinting revealed conserved potential binding sites for the MZF-1 transcription factor in both upstream promoter and intron/intron 1, and for the MEF2, MyTI, Oct-1 and NFAT transcription factors in the intron(s). The presence of a conserved NFAT-binding site and CPE indicates involvement of PrPC in signal transduction and synaptic plasticity. (c) 2004 Elsevier B.V. All rights reserved.

Human SULT1A genes: Cloning and activity assays of the SULT1A promoters

The three human SULT1A sulfotransferase enzymes are closely related in amino acid sequence (>90%), yet differ in their substrate preference and tissue distribution. SULT1A1 has a broad tissue distribution and metabolizes a range of xenobiotics as well as endogenous substrates such as estrogens and iodothyronines. While the localization of SULT1A2 is poorly understood, it has been shown to metabolize a number of aromatic amines. SULT1A3 is the major catecholamine sulfonating form, which is consistent with it being expressed principally in the gastrointestinal tract. SULT1A proteins are encoded by three separate genes, located in close proximity to each other on chromosome 16. The presence of differential 5′-untranslated regions identified upon cloning of the SULT1A cDNAs suggested the utilization of differential transcriptional start sites and/or differential splicing. This chapter describes the methods utilized by our laboratory to clone and assay the activity of the promoters flanking these different untranslated regions found on SULT1A genes. These techniques will assist investigators in further elucidating the differential mechanisms that control regulation of the human SULT1A genes. They will also help reveal how different cellular environments and polymorphisms affect the activity of SULT1A gene promoters.

Physical and psychological factors maintain long-term predictive capacity post-whiplash injury

Higher initial levels of pain and disability, older age, cold hyperalgesia, impaired sympathetic vasoconstriction and moderate post-traumatic stress symptoms have been shown to be associated with poor outcome 6 months following whiplash injury. This study prospectively investigated the predictive capacity of these variables at a long-term follow-up. Sixty-five of an initial cohort of 76 acutely injured whiplash participants were followed to 2-3 years post-accident. Motor function (ROM; kinaesthetic sense; activity of the superficial neck flexors (EMG) during cranio-cervical flexion), quantitative sensory testing (pressure, thermal pain thresholds and brachial plexus provocation test), sympathetic vasoconstrictor responses and psychological distress (GHQ-28, TSK and IES) were measured. The outcome measure was Neck Disability Index (NDI) scores. Participants with ongoing moderate/severe symptoms at 2-3 years continued to manifest decreased ROM, increased EMG during cranio-cervical flexion, sensory hypersensitivity and elevated levels of psychological distress when compared to recovered participants and those with milder symptoms. The latter two groups showed only persistent deficits in cervical muscle recruitment patterns. Higher initial NDI scores (OR 1.00-1.1), older age (OR 1.00-1.13), cold hyperalgesia (OR 1.1-1.13) and post-traumatic stress symptoms (OR 1.03-1.2) remained significant predictors of poor outcome at long-term follow-up (r(2) = 0.56). The robustness of these physical and psychological factors suggests that their assessment in the acute stage following whiplash injury will be important. (c) 2006 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.

Neuronal voltage-gated sodium channel subtypes: Key roles in inflammatory and neuropathic pain

Voltage-gated sodium channels (VGSCs) play an important role in neuronal excitability. Regulation of VGSC activity is a complex phenomenon that occurs at multiple levels in the cell, including transcriptional regulation, post-translational modification and membrane insertion and retrieval. Multiple VGSC subtypes exist that vary in their biophysical and pharmacological properties and tissue distribution. Any alteration of the VGSC subtype profile of a neuron or the mechanisms that regulate VGSC activity can cause significant changes in neuronal excitability. Inflammatory and neuropathic pain states are characterised by alterations in VGSC subtype composition and activity in sensory neurons. This review focuses on the VGSC subtypes involved in such pain states. (c) 2006 Elsevier Ltd. All rights reserved.