Does the neuroprotective role of anandamide display diurnal variations?

The endocannabinoid system is a component of the neuroprotective mechanisms that an organism displays after traumatic brain injury (TBI). A diurnal variation in several components of this system has been reported. This variation may influence the recovery and survival rate after TBI. We have previously reported that the recovery and survival rate of rats is higher if TBI occurs at 1:00 than at 13:00. This could be explained by a diurnal variation of the endocannabinoid system. Here, we describe the effects of anandamide administration in rats prior to the induction of TBI at two different times of the day: 1:00 and 13:00. We found that anandamide reduced the neurological damage at both times. Nevertheless, its effects on bleeding, survival, food intake, and body weight were dependent on the time of TBI. In addition, we analyzed the diurnal variation of the expression of the cannabinoid receptors CB1R and CB2R in the cerebral cortex of both control rats and rats subjected to TBI. We found that CB1R protein was expressed more during the day, whereas its mRNA level was higher during the night. We did not find a diurnal variation for the CB2R. In addition, we also found that TBI increased CB1R and CB2R in the contralateral hemisphere and disrupted the CB1R diurnal cycle.

Evaluation of real-time polymerase chain reaction for the diagnosis of protozoal myeloencephalitis in horses using cerebrospinal fluid.

BACKGROUND: Equine protozoal myeloencephalitis (EPM) caused by Sarcocystis neurona remains an antemortem diagnostic challenge in some horses. Recent work suggested the use of real-time PCR (rtPCR) on cerebrospinal fluid (CSF) as a promising diagnostic tool. OBJECTIVE: To evaluate the sensitivity and specificity of S. neurona rtPCR on CSF for EPM diagnosis using horses with EPM and S. neurona-seropositive horses with other neurologic conditions. ANIMALS: Ninety-nine horses with neurologic disease that underwent complete neurologic examination, CSF collection, and, if euthanized, necropsy including the central nervous system (CNS). METHODS: Retrospective case-control study using banked CSF samples. Samples from horses with neurologic abnormalities and necropsy-confirmed EPM diagnosis, presumptive EPM diagnosis using strict criteria (SnSAG2/4/3 ELISA serum:CSF titer ratios <50) and horses diagnosed with other neurologic diseases were used. RESULTS: Fifty-two horses had EPM; 23 were confirmed on necropsy, and 29 were presumptive clinical diagnoses. The other 47 horses all had necropsy-confirmed diagnoses. Four of the 47 horses had normal neurologic findings on necropsy and the remaining 43 horses had neurologic diseases including equine degenerative myeloencephalopathy (EDM), cervical vertebral stenotic myelopathy, trauma, and other miscellaneous conditions. One CSF sample was weakly positive for S. neurona by rtPCR, this sample was obtained from a horse with confirmed EDM. Samples from the other 98 horses were negative for S. neurona by rtPCR. CONCLUSIONS AND CLINICAL IMPORTANCE: Our study contradicts previous conclusions that S. neurona rtPCR is potentially useful for EPM diagnosis, because our results indicate that the assay has a low sensitivity (0%) for EPM.

Protein kinase C mediated inhibition of endothelial L-arginine transport is mediated by MARCKS protein.

The endothelium plays a vital role in the maintenance of vascular tone and structural vascular integrity, principally mediated via the actions of nitric oxide (NO). L-arginine is the immediate substrate for NO synthesis, and the availability of extracellular L-arginine is critical for the production of NO. Activation of protein kinase C (PKC) dependent signalling pathways are a feature of a number of cardiovascular disease states, and in this study we aimed to systematically evaluate the mechanism by which PKC regulates L-arginine transport in endothelial cells. In response to PKC activation (PMA 100 nM, 30 min), [(3)H]L-arginine uptake by bovine aortic endothelial cells (BAEC) was reduced to 45+4% of control (p<0.05). This resulted from a 53% reduction in the Vmax (p<0.05), with no change in the K(m) for L-arginine. Western blot analysis and confocal microscopy revealed no change in the expression or membrane distribution of CAT-1, the principal BAEC L-arginine transporter. Moreover in (32)P-labeling studies, PMA exposure did not result in CAT-1 phosphorylation. We therefore explored the possibility that PKC altered and interaction with MARCKS protein, a candidate membrane associated protein. By co-immunoprecipitation we show that CAT-1 interacts with, a membrane associated protein, that was significantly inhibited by PKC activation (p<0.05). Moreover antisense inhibition of MARCKS abolished the PMA effect on L-arginine transport. PKC dependent mechanisms regulate the transport of L-arginine, mediated via process involving MARCKS.

Association Between Pain Sensitivity and Endogenous Supraspinal Pain Modulation in Patients with Orofacial Pain.

Address correspondence to: Dr. Carla S. Enriquez, Physical Therapy Program, Stockton University School of Health Sciences, 101 Vera King Farris Drive, Galloway, NJ 08205-9441, USA. Tel 609-626-3508, fax 609-652-4858. carla.enriquez@stockton.edu. PURPOSE: To investigate the association between pain sensitivity and endogenous supraspinal pain modulation activity using quantitative sensory tests (QST) in patients with orofacial pain. METHODS: Temporal summation (TS) testing and conditioned pain modulation (CPM) are QSTs used to quantify individual pain processing mechanisms. TS testing was used to assess pain sensitivity which correlates to central sensitization and CPM for endogenous pain modulation. These tests were administered to a convenience sample of patients with orofacial pain (n=40) seen for initial examination in an orofacial pain clinic. Bivariate correlation analyses were conducted on TS and CPM scores, including age and gender. RESULTS: A strong linear correlation was found between pain sensitivity and endogenous pain modulation (r=0.69, p<0.001). QST outcomes suggest that inefficient endogenous pain modulation is positively correlated with the magnitude of pain sensitivity among individuals who seek treatment for orofacial pain. A weak linear correlation was also found between TS and age (r=0.37, p=0.02). CONCLUSION: QSTs demonstrated a strong association between heightened pain sensitivity and inefficient endogenous pain modulation revealed from higher pain report on TS and CPM testing in patients with orofacial pain. Our findings provide supporting evidence on the impairments in pain-processing mechanisms that require consideration when evaluating orofacial pain etiology, where increased pain sensitivity and aberrant pain modulation appear to significantly contribute in prolonged and persistent pain experience.

17 beta-hydroxysteroid dehydrogenase type XI localizes to human steroidogenic cells.

We searched expressed sequence tag databases with conserved domains of the short-chain alcohol dehydrogenase superfamily and identified another isoform of 17 beta-hydroxysteroid dehydrogenase, 17 beta HSDXI. This enzyme converts 5 alpha-androstane-3 alpha, 17 beta-diol to androsterone. The substrate has been implicated in supporting gestation and modulating gamma-aminobutyric acid receptor activity. 17 beta HSDXI is colinear with human retinal short-chain dehydrogenase/reductase retSDR2, a protein with no known biological activity (accession no. AAF06939). Of the proteins with known function, 17 beta HSDXI is most closely related to the retinol-metabolizing enzyme retSDR1, with which it has 30% identity. There is a polymorphic stretch of 15 adenosines in the 5' untranslated region of the cDNA sequence and a silent polymorphism at C719T. A 17 beta HSDXI construct with a stretch of 20 adenosines was found to produce significantly more enzyme activity than constructs containing 15 or less adenosines (43% vs. 26%, P < 0.005). The C719T polymorphism is present in 15% of genomic DNA samples. Northern blot analysis showed high levels of 17 beta HSDXI expression in the pancreas, kidney, liver, lung, adrenal, ovary, and heart. Immunohistochemical staining for 17 beta HSDXI is strong in steroidogenic cells such as syncytiotrophoblasts, sebaceous gland, Leydig cells, and granulosa cells of the dominant follicle and corpus luteum. In the adrenal 17 beta HSDXI, staining colocalized with the distribution of 17 alpha-hydroxylase but was stronger in the mid to outer cortex. 17 beta HSDXI was also found in the fetus and increased after birth. Liver parenchymal cells and epithelium of the endometrium and small intestine also stained. Regulation studies in mouse Y1 cells showed that cAMP down-regulates 17 beta HSDXI enzymatic activity (40% vs. 32%, P < 0.05) and reduces gene expression to undetectable levels. All-trans-retinoic acid did not affect 17 beta HSDXI expression or activity, but addition of the retinoid together with cAMP significantly decreased activity over cAMP alone (32% vs. 23%, P < 0.05). Cloning and sequencing of the 17 beta HSDXI promoter identified the potential nuclear receptor steroidogenic factor-1 half-site TCCAAGGCCGG, and a cluster of three other potential steroidogenic factor-1 half-sites were found in the distal part of intron 1. Collectively, these results suggest a role for 17 beta HSDXI in androgen metabolism during steroidogenesis and a possible role in nonsteroidogenic tissues including paracrine modulation of 5 alpha-androstane-3 alpha, 17 beta-diol levels. 17 beta HSDXI could act by metabolizing compounds that stimulate steroid synthesis and/or by generating metabolites that inhibit it.