Localisation of NMU1R and NMU2R in human and rat central nervous system and effects of neuromedin-U following central administration in rats

Rationale: Neuromedin-U (NmU) is an agonist at NMU1R and NMU2R. The brain distribution of NmU and its receptors, in particular NMU2R, suggests widespread central roles for NmU. In agreement, centrally administered NmU affects feeding behaviour, energy expenditure and pituitary output. Further central nervous system (CNS) roles for NmU warrant investigation. Objectives: To investigate the CNS role of NmU by mapping NMU1R and NMU2R mRNA and measuring the behavioural, endocrine, neurochemical and c-fos response to intracerebroventricular (i.c.v.) NmU. Methods: Binding affinity and functional potency of rat NmU was determined at human NMU1R and NMU2R. Expression of NMU1R and NMU2R mRNA in rat and human tissue was determined using semi-quantitative reverse-transcription polymerase chain reaction. In in-vivo studies, NmU was administered i.c.v. to male Sprague-Dawley rats, and changes in grooming, motor activity and pre-pulse inhibition (PPI) were assessed. In further studies, plasma endocrine hormones, [DOPAC + HVA]/[dopamine] and [5-HIAA]/[5-HT] ratios and levels of Fos-like immunoreactivity (FLI) were measured 20 min post-NmU (i.c.v.). Results: NmU bound to NMU1R (KI, 0.11±0.02 nM) and NMU2R (KI, 0.21±0.05 nM) with equal affinity and was equally active at NMU1R (EC50, 1.25±0.05 nM) and NMU2R (EC50, 1.10±0.20 nM) in a functional assay. NMU2R mRNA expression was found at the highest levels in the CNS regions of both rat and human tissues. NMU1R mRNA expression was restricted to the periphery of both species with the exception of the rat amygdala. NmU caused a marked increase in grooming and motor activity but did not affect PPI. Further, NmU decreased plasma prolactin but did not affect levels of corticosterone, luteinising hormone or thyroid stimulating hormone. NmU elevated levels of 5-HT in the frontal cortex and hypothalamus, with decreased levels of its metabolites in the hippocampus and hypothalamus, but did not affect dopamine function. NmU markedly increased FLI in the nucleus accumbens, frontal cortex and central amygdala. Conclusions: These data provide further evidence for widespread roles for NmU and its receptors in the brain

LDL Receptor Knock-Out Mice Are a Physiological Model Particularly Vulnerable to Study the Onset of Inflammation in Non-Alcoholic Fatty Liver Disease

Non-alcoholic steatohepatitis (NASH) involves steatosis combined with inflammation, which can progress into fibrosis and cirrhosis. Exploring the molecular mechanisms of NASH is highly dependent on the availability of animal models. Currently, the most commonly used animal models for NASH imitate particularly late stages of human disease. Thus, there is a need for an animal model that can be used for investigating the factors that potentiate the inflammatory response within NASH. We have previously shown that 7-day high-fat-high-cholesterol (HFC) feeding induces steatosis and inflammation in both APOE2ki and Ldlr(-/-) mice. However, it is not known whether the early inflammatory response observed in these mice will sustain over time and lead to liver damage. We hypothesized that the inflammatory response in both models is sufficient to induce liver damage over time.APOE2ki and Ldlr(-/-) mice were fed a chow or HFC diet for 3 months. C57Bl6/J mice were used as control.Surprisingly, hepatic inflammation was abolished in APOE2ki mice, while it was sustained in Ldlr(-/-) mice. In addition, increased apoptosis and hepatic fibrosis was only demonstrated in Ldlr(-/-) mice. Finally, bone-marrow-derived-macrophages of Ldlr(-/-) mice showed an increased inflammatory response after oxidized LDL (oxLDL) loading compared to APOE2ki mice.Ldlr(-/-) mice, but not APOE2ki mice, developed sustained hepatic inflammation and liver damage upon long term HFC feeding due to increased sensitivity for oxLDL uptake. Therefore, the Ldlr(-/-) mice are a promising physiological model particularly vulnerable for investigating the onset of hepatic inflammation in non-alcoholic steatohepatitis

Identification of the calcitonin receptor in osteoarthritic chondrocytes

<p>Abstract</p> <p>Background</p> <p>Preclinical and clinical studies have shown that salmon calcitonin has cartilage protective effects in joint degenerative diseases, such as osteoarthritis (OA). However, the presence of the calcitonin receptor (CTR) in articular cartilage chondrocytes is yet to be identified. In this study, we sought to further investigate the expression of the CTR in naïve human OA articular chondrocytes to gain further confirmation of the existents of the CTR in articular cartilage.</p> <p>Methods</p> <p>Total RNA was purified from primary chondrocytes from articular cartilage biopsies from four OA patients undergoing total knee replacement. High quality cDNA was produced using a dedicated reverse transcription polymerase chain reaction (RT-PCR) protocol. From this a nested PCR assay amplifying the full coding region of the CTR mRNA was completed. Western blotting and immunohistochemistry were used to characterize CTR protein on protein level in chondrocytes.</p> <p>Results</p> <p>The full coding transcript of the CTR isoform 2 was identified in all four individuals. DNA sequencing revealed a number of allelic variants of the gene including two potentially novel polymorphisms: a frame shift mutation, +473del, producing a shorter form of the receptor protein, and a single nucleotide polymorphism in the 3' non coding region of the transcript, +1443 C>T. A 53 kDa protein band, consistent with non-glycosylated CTR isoform 2, was detected in chondrocytes with a similar size to that expressed in osteoclasts. Moreover the CTR was identified in the plasma membrane and the chondrocyte lacuna of both primary chondrocytes and OA cartilage section.</p> <p>Conclusions</p> <p>Human OA articular cartilage chondrocytes do indeed express the CTR, which makes the articular a pharmacological target of salmon calcitonin. In addition, the results support previous findings suggesting that calcitonin has a direct anabolic effect on articular cartilage.</p

LDLR Expression and Localization Are Altered in Mouse and Human Cell Culture Models of Alzheimer's Disease

Alzheimer's disease (AD) is a chronic neurodegenerative disorder and the most common form of dementia. The major molecular risk factor for late-onset AD is expression of the ε-4 allele of apolipoprotein E (apoE), the major cholesterol transporter in the brain. The low-density lipoprotein receptor (LDLR) has the highest affinity for apoE and plays an important role in brain cholesterol metabolism.Using RT-PCR and western blotting techniques we found that over-expression of APP caused increases in both LDLR mRNA and protein levels in APP transfected H4 neuroglioma cells compared to H4 controls. Furthermore, immunohistochemical experiments showed aberrant localization of LDLR in H4-APP neuroglioma cells, Aβ-treated primary neurons, and in the PSAPP transgenic mouse model of AD. Finally, immunofluorescent staining of LDLR and of γ- and α-tubulin showed a change in LDLR localization preferentially away from the plasma membrane that was paralleled by and likely the result of a disruption of the microtubule-organizing center and associated microtubule network.These data suggest that increased APP expression and Aβ exposure alters microtubule function, leading to reduced transport of LDLR to the plasma membrane. Consequent deleterious effects on apoE uptake and function will have implications for AD pathogenesis and/or progression

The effect of entrepreneurial market orientation on firm performance: The case of SMEs in Egypt

[abstract not available