Toll-like receptor 5 knock-out mice exhibit a specific low level of anxiety.

While toll-like receptors (TLRs), which mediate innate immunity, are known to play an important role in host defense, recent work suggest their involvement in some integrated behaviors, including anxiety, depressive and cognitive functions. Here, we investigated the potential involvement of the flagellin receptor, TLR5, in anxiety, depression and cognitive behaviors using male TLR5 knock-out (KO) mice. We aobserved a specific low level of basal anxiety in TLR5 KO mice with an alteration of the hypothalamo-pituitary axis (HPA) response to acute restraint stress, illustrated by a decrease of both plasma corticosterone level and c-fos expression in the hypothalamic paraventricular nucleus where TLR5 was expressed, compared to WT littermates. However, depression and cognitive-related behaviors were not different between TLR5 KO and WT mice. Nor there were significant changes in the expression of some cytokines (IL-6, IL-10 and TNF-α) and other TLRs (TLR2, TLR3 and TLR4) in the prefrontal cortex, amygdala and hippocampus of TLR5 KO mice compared to WT mice. Moreover, mRNA expression of BDNF and glucocorticoid receptors in the hippocampus and amygdala, respectively, was not different. Finally, acute intracerebroventricular administration of flagellin, a specific TLR5 agonist, or chronic neomycin treatment did not exhibit a significant main effect, only a significant main effect of genotype was observed between TLR5 KO and WT mice. Together, those findings suggest a previously undescribed and specific role of TLR5 in anxiety and open original prospects in our understanding of the brain-gut axis function.

Colonic overexpression of the T-type calcium channel Cav 3.2 in a mouse model of visceral hypersensitivity and in irritable bowel syndrome patients.

BACKGROUND: Among the different mechanisms involved in irritable bowel syndrome (IBS) physiopathology, visceral hypersensitivity seems to play a key role. It involves sensitization of the colonic primary afferent fibers, especially through an overexpression of ion channels. The aims of this translational study were to investigate the colonic expression of Cav 3.2 calcium channels and their involvement in an animal model of colonic hypersensitivity, and to assess their expression in the colonic mucosa of symptomatic IBS patients. METHODS: This bench-to-bed study combined a preclinical experimental study on mice and a case-control clinical study. Preclinical studies were performed on wild-type and Cav 3.2-KO mice. Colonic sensitivity and Cav 3.2 expression were studied after a low-dose treatment of dextran sodium sulfate (DSS 0.5%). Regarding the clinical study, colonic biopsies were performed in 14 IBS patients and 16 controls during a colonoscopy to analyze the mucosal Cav 3.2 expression. KEY RESULTS: Wild-type, but not Cav 3.2-KO, mice developed visceral hypersensitivity without colonic inflammation, after 0.5% DSS treatment. A significant increase of Cav 3.2 mRNA (p = 0.04) was found in the colon of low-dose DSS-treated wild-type (WT) mice compared to their controls. In human colonic biopsies, the Cav 3.2 mRNA level was significantly higher in the IBS group compared to the control group (p = 0.01). The immunofluorescence staining revealed their protein expression in colonic mucosa, particularly in nerve fibers. CONCLUSIONS & INFERENCES: This translational study supports the involvement of the calcium channels Cav 3.2 in abdominal pain, as observed in IBS patients. It opens new therapeutic perspectives based on molecules specifically blocking these channels.

Recovery of gel-separated proteins for in-solution digestion and mass spectrometry.

A protocol for mass spectrometry of gel-separated proteins resulting in significantly increased sequence coverage and in improved possibilities for detection and identification of posttranslational modifications was developed. In relation to the standard in-gel digestion procedure, the sequence coverage using a combination of matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry was on the average increased by 30%. The method involves electroblotting of the gel-separated proteins to a poly(vinylidene difluoride) membrane. The proteins are extracted from the membrane using a solution of 1% trifluoroacetic acid in 70% acetonitrile and lyophilized. After reconstitution of the protein extract in digestion buffer, proteolytic cleavage is carried out in-solution as opposed to the standard in-gel digestion procedure. This allows recovery of large and hydrophobic peptides for mass spectrometry and reduces the risk for entrapment of proteolytic peptides in the gel matrix. The method was applied to proteins in the 30-40-kDa range with highly different structural properties. The improved ability to localize and determine protein modifications is shown for N-terminal acetylation and methylation of a histidine residue. Furthermore, the method enables fast screening of homologous protein sequences.

Electron tomography reveals posttranscriptional binding of pre-mRNPs to specific fibers in the nucleoplasm.

Using electron tomography, we have analyzed whether the Balbiani ring (BR) pre-mRNP particles in transit from the gene to the nuclear pore complex (NPC) are bound to any structure that could impair free diffusion through the nucleoplasm. We show that one-third of the BR particles are in contact with thin connecting fibers (CFs), which in some cases merge into large fibrogranular clusters. The CFs have a specific protein composition different from that of BR particles, as shown by immuno-EM. Moreover, we have identified hrp65 as one of the protein components of the CFs. The sequencing of hrp65 cDNA reveals similarities with hnRNP proteins and splicing factors. However, hrp65 is likely to have a different function because it does not bind to nascent pre-mRNA and is not part of the pre-mRNP itself. Taken together, our observations indicate that pre-mRNPs are not always freely diffusible in the nucleoplasm but interact with fibers of specific structure and composition, which implies that some of the posttranscriptional events that the pre-mRNPs undergo before reaching the NPC occur in a bound state.

The hrp23 protein in the balbiani ring pre-mRNP particles is released just before or at the binding of the particles to the nuclear pore complex.

Balbiani ring (BR) pre-mRNP particles reside in the nuclei of salivary glands of the dipteran Chironomus tentans and carry the message for giant-sized salivary proteins. In the present study, we identify and characterize a new protein component in the BR ribonucleoprotein (RNP) particles, designated hrp23. The protein with a molecular mass of 20 kD has a single RNA-binding domain and a glycine-arginine-serine-rich auxiliary domain. As shown by immunoelectron microscopy, the hrp23 protein is added to the BR transcript concomitant with transcription, is still present in the BR particles in the nucleoplasm, but is absent from the BR particles that are bound to the nuclear pore complex or are translocating through the central channel of the complex. Thus, hrp23 is released just before or at the binding of the particles to the nuclear pore complex. It is noted that hrp23 behaves differently from two other BR RNP proteins earlier studied: hrp36 and hrp45. These proteins both reach the nuclear pore complex, and hrp36 even accompanies the RNA into the cytoplasm. It is concluded that each BR RNA-binding protein seems to have a specific flow pattern, probably related to the particular role of the protein in gene expression.

Iodide and T4 kinetics in plasma, thyroid gland and skin of 10-day-old rats: effects of iodine deficiency.

The effects of iodine deficiency on the peripheral metabolism of thyroid hormone in immature rat were evaluated by measuring the kinetics of iodide and thyroxine (T4) in control and iodine-deficient 10-day-old rats. Iodine-deficient pups were obtained by giving the mother drinking water containing perchlorate; this anion is not transferred but prevents iodine transfer in the mother's milk. Labelled iodocompounds were measured in plasma, thyroid and skin for 48 h following intravenous injection of Na131I plus [125I]T4. Data were interpreted by compartmental analysis. The iodide plasma clearance rate, plasma equivalent distribution volume, plasma concentration, production and iodine thyroid content of iodine-deficient rats were significantly lower (-29%, -31%, -84%, -89% and -87% respectively) than in control 10-day-old rats. The iodide thyroid uptake was reduced (-47%) but remained higher than the release of iodine as T4. Cutaneous iodine was lost much more quickly by iodine-deficient pups than by control pups, explaining the decreased iodide distribution volume. The parameters of T4 metabolism were not changed by iodine deficiency, except for a slight but significant reduction of the thyroxinemia and T4 pools (-13%). Thence, T4 production was not significantly changed (about 8 pmol/h in control and iodine-deficient rats). The labelled T4 curves in iodine-deficient and control skin were superimposed and the patterns of labelled T3 derived from [125I]T4 were identical. Thus, the skin of immature rats converts T4 to T3; this process was not disturbed by iodine deficiency. The thyroid function of immature rats is particularly resistant to iodine deficiency, but the mechanisms remain unknown.