Psychosine-induced alterations in peroxisomes of twitcher mouse liver

Krabbe disease is a neuroinflammatory disorder in which galactosylsphingosine (psychosine) accumulates in nervous tissue. To gain insight into whether the psychosine-induced effects in nervous tissue extend to peripheral organs, we investigated the expression of cytokines and their effects on peroxisomal structure/functions in twitcher mouse liver (animal model of Krabbe disease). Immunofluorescence analysis demonstrated TNF-α and IL-6 expression, which was confirmed by mRNAs quantitation. Despite the presence of TNF-α, lipidomic analysis did not indicate a significant decrease in sphingomyelin or an increase in ceramide fractions. Ultrastructural analysis of catalase-dependent staining of liver sections showed reduced reactivity without significant changes in peroxisomal contents. This observation was confirmed by assaying catalase activity and quantitation of its mRNA, both of which were found significantly decreased in twitcher mouse liver. Western blot analysis demonstrated a generalized reduction of peroxisomal matrix and membrane proteins. These observations indicate that twitcher mouse pathobiology extends to the liver, where psychosine-induced TNF-α and IL-6 compromise peroxisomal structure and functions.     

Inhibition of forebrain μ-opioid receptor signaling by low concentrations of rimonabant does not require cannabinoid receptors and directly involves μ-opioid receptors

Increasing number of publications shows that cannabinoid receptor 1 (CB(1)) specific compounds might act in a CB(1) independent manner, including rimonabant, a potent CB(1) receptor antagonist. Opioids, cannabinoids and their receptors are well known for their overlapping pharmacological properties. We have previously reported a prominent decrease in μ-opioid receptor (MOR) activity when animals were acutely treated with the putative endocannabinoid noladin ether (NE). In this study, we clarified whether the decreased MOR activation caused by NE could be reversed by rimonabant in CB(1) receptor deficient mice. In functional [(35)S]GTPγS binding assays, we have elucidated that 0.1mg/kg of intraperitoneal (i.p.) rimonabant treatment prior to that of NE treatment caused further attenuation on the maximal stimulation of Tyr-d-Ala-Gly-(NMe)Phe-Gly-ol (DAMGO), which is a highly specific MOR agonist. Similar inhibitory effects were observed when rimonabant was injected i.p. alone and when it was directly applied to forebrain membranes. These findings are cannabinoid receptor independent as rimonabant caused inhibition in both CB(1) single knockout and CB(1)/CB(2) double knockout mice. In radioligand competition binding assays we highlighted that rimonabant fails to displace effectively [(3)H]DAMGO from MOR in low concentrations and is highly unspecific on the receptor at high concentrations in CB(1) knockout forebrain and in their wild-type controls. Surprisingly, docking computational studies showed a favorable binding position of rimonabant to the inactive conformational state of MOR, indicating that rimonabant might behave as an antagonist at MOR. These findings were confirmed by radioligand competition binding assays in Chinese hamster ovary cells stably transfected with MOR, where a higher affinity binding site was measured in the displacement of the tritiated opioid receptor antagonist naloxone. However, based on our in vivo data we suggest that other, yet unidentified mechanisms are additionally involved in the observed effects.     

The individuality of mice

Mutant mice simulating human CNS disorders are used as models for therapeutic drug development. Drug evaluation requires a coherent correlation between behavioral phenotype and drug status. Variations in behavioral responses could mask such correlations, a problem highlighted by the three-site studies of Crabbe et al. (1999) and Wahlsten et al. (2003a). Factors contributing to variation are considered, focusing on differences between individual animals. Genetic differences due to minisatellite variation suggest that each mouse is genetically distinct. Effects during gestation, including maternal stress, influence later life behavior; while endocrine exchanges between fetus and parent, and between male and female fetuses dependent on intrauterine position, also contribute. Pre and perinatal nutrition and maternal attention also play a role. In adults, endocrine cyclicity in females is a recognized source of behavioral diversity. Notably, there is increasing recognition that groups of wild and laboratory mice have complex social structures, illustrated through consideration of Crowcroft (1966). Dominance status can markedly modify behavior in test paradigms addressing anxiety, locomotion and aggressiveness, to an extent comparable to mutation or drug status. Understanding how such effects amplify the behavioral spectrum displayed by otherwise identical animals will improve testing.     

Heparan sulfate 2-O-sulfotransferase (Hs2st) and mouse development

Heparan sulphate 2-O-sulphotransferase (Hs2st) acts at an intermediate stage in the pathway of biosynthesis of heparan sulphate (HS), catalysing the transfer of sulphate from 3-phosphoadenosine-5-phosphosulfate (PAPS) to the C2-position of selected hexuronic acid residues within the maturing HS chain. It is well established that 2-O-sulphation within HS, particularly of iduronate residues, is essential for HS to participate in a variety of high-affinity ligand-binding interactions. HS plays a central role in embryonic development and cellular function, modulating the activities of an extensive range of growth factors. Interestingly, in contrast to the early failure of embryos entirely lacking HS, Hs2st ^–/– mice survive until birth, but die perinatally due to a complete failure of kidney formation. The phenotype of Hs2st ^–/– mutant kidneys suggests that signalling between two tissues, ureteric bud and metanephric mesenchyme, is disrupted. We discuss candidate signalling molecules that may mediate this interaction. The HS generated by these mice lacks 2-O-sulphate groups but is extensively modified above wild type levels by O-sulphation at C-6 of glucosamine-N-sulfate (GlcNS) residues. We will discuss the potentially altered role of this atypical HS in growth factor signalling. Published in 2003.     

Opioid receptor-like 1 (ORL1) receptor binding and the biological properties of Ac-Arg-Tyr-Tyr-Arg-Ile-Arg-NH2 and its analogs.

Hexapeptides such as Ac-Arg-Tyr-Tyr-Arg-Ile-Lys-NH(2) and Ac-Arg-Tyr-Tyr-Arg-Trp-Arg-NH(2) have been isolated from a combinatorial peptide library as small peptide ligands for the opioid peptide-like 1 (ORL1) receptor. To investigate the detailed structural requirements of hexapeptides, 25 analogs of these hexapeptides, based on the novel analog Ac-Arg-Tyr-Tyr-Arg-Ile-Arg-NH(2) (1), were synthesized and tested for their ORL1 receptor affinity and agonist/antagonist activity on mouse vas deferens (MVD) tissues. Analog 1 and its Cit(6)-analog (10) were found to possess high affinity to the ORL1 receptor, comparable to that of nociceptin/orphanin FQ, and exhibited potent antagonist activity (pA(2) values of 7.77 for 1 and 7.51 for 10, which are higher than that of [NPhe(1)]nociceptin(1-13)-NH(2) (6.90) on MVD assay. It was also found that the amino acid residue in position 5 plays a key role in agonist/antagonist activity, i.e. an L-configuration aliphatic amino acid is required for potent antagonist activity, while a nonchiral or D-configuration residue produces potent agonist activity. These lines of evidence may provide insight into the mechanisms controlling agonist/antagonist switching in the ORL1 receptor, and may also serve to help developing more potent ORL1 agonists and antagonists.     

Amplification of evoked potentials recorded from mouse hippocampal slices by very low repetition rate pulsed magnetic fields

The influence of low repetition rate pulsed magnetic fields (LRMF) on the evoked potential (population spike) recorded from mouse hippocampal slices was investigated. LRMF were applied according to two protocols. In protocol A, LRMF applied with a constant strength (15 mT) and frequency ranging from 0.03 to 0.5 Hz resulted in an amplification of the potential. Although the frequency of 0.16 Hz was the most effective, enhancing the population spike by over 280%, it also caused an increase in spontaneous activity, seizures, and cessation of neuronal activity in 50% of the slices. In protocol B, LRMF were applied with a variable intensity (9-15 mT) and in cycles of different duration ranging from 5 to 20 min. While an increase in the amplitude of the population spike was observed in all slices exposed to LRMF applied according to protocol B, the longest exposure was the most effective. Neither seizures nor an increase in the spontaneous activity were observed in this group of the slices. These results support and extend our previous data and characterize further the relation between the pattern of applied magnetic fields and their influence on the nervous system.     

Studies on new,centrally active and reversible acetylcholinesterase inhibitors

We have synthesized the tertiary amines of pyridostigmine and neostigmine, 3-pyridinol dimethylcarbamate (norpyridostigmine) and 3-dimethylaminophenol dimethylcarbamate (norneostigmine) respectively, and we have tested their abilities to cross the blood-brain barrier and inhibit mouse brainAChE activity. The in vivo inhibition of AChE activity by norpyridostigmine reaches 72% at 10 minutes which is comparable to that seen with physostigmine (73% at 10 minutes). Inhibition by norneostigmine is less effective (50% at 10 minutes) and approaches that obtained with tetrahydroaminoacridine (57% at 10 minutes). These data show that both norpyridostigmine and norneostigmine cross the blood-brain barrier and that they are effective inhibitors of mouse brain AChE activity. These drugs could be useful in the treatment of memory, impairment associated with Alzheimer's disease, and other memory disorders.     

A phenotypic and molecular characterization of the fmr1-tm1Cgr fragile X mouse

Fragile X Syndrome is the most common form of inherited mental retardation. It is also known for having a substantial behavioral morbidity, including autistic features. In humans, Fragile X Syndrome is almost always caused by inactivation of the X-linked FMR1 gene. A single knockout mouse model, fmr1-tm1Cgr, exists. In this report we further characterize the cognitive and behavioral phenotype of the fmr1-tm1Cgr Fragile X mouse through the use of F1 hybrid mice derived from two inbred strains (FVB/NJ and C57BL/6J). Use of F1 hybrids allows focus on the effects of the fmr1-tm1Cgr allele with reduced influence from recessive alleles present in the parental inbred strains. We find that the cognitive phenotype of fmr1-tm1Cgr mice, including measures of working memory and learning set formation that are known to be seriously impacted in humans with Fragile X Syndrome, are essentially normal. Further testing of inbred strains supports this conclusion. Thus, any fmr1-tm1Cgr cognitive deficit is surprisingly mild or absent. There is, however, clear support presented for a robust audiogenic seizure phenotype in all strains tested, as well as increased entries into the center of an open field. Finally, a molecular examination of the fmr1-tm1Cgr mouse shows that, contrary to common belief, it is not a molecular null. Implications of this finding for interpretation of the phenotype are discussed.     

M16添加牛磺酸和EDTA支持昆明白小鼠体外受精并发育至囊胚

在以往研究工作的基础上证明了通过添加2.5mmol/L的牛磺酸和0.1mmol/L的EDTA至M16培养液中,可支持昆明白小鼠的体外受精（IVF）,并支持受精卵突破2－细胞阻滞发育至囊胚期。本研究进一步证明了牛磺酸和EDTA在昆明小鼠早期胚胎发育和克服2－细胞阻滞中起关键作用。 Abstract:The results of our early experiments show that the 2-cell block can be overcome by culturing zygote in modified M16 ,modified CZB and TE medium.Our research shows that the taurine and EDTA play key role in overcoming 2-cell block in Kunming mouse.The results show that the addition of taurine and EDTA to M16 medium can support the IVF and develoment to blastocyst in vitro in Kunming strain mice.This is the first report that M16 medium added with taurine and EDTA can be used in both IVF and culture medium to overcome the 2-cell block of embryo development in Kunming strain mouse.