Odorant receptor expression in the mouse cerebral cortex

Mammalian odorant receptors have been known to be involved not only in odorant detection but also in neuronal development of olfactory sensory neurons. We have examined a possibility of odorant receptor expression in nonolfactory neurons in the mouse. Mouse odorant receptors (M71, C6, and OR3), two of which were already shown to be functionally activated by odorants in heterologous systems, were detected by polymerase chain reactions (PCRs) from the cerebral cortex but not from other brain tissues. Degenerate PCR further suggested that other odorant receptors were also expressed in the mouse cerebral cortex. One of these receptors showed high sequence-match with a putative chick odorant receptor OR7 transiently expressed in the notochord during development. In situ hybridization detected signals for M71 and C6 receptors in the layer II cortical pyramidal neurons located in the occipital pole. In the M71-IRES-tauLacZ mouse, in which M71 expression was genetically marked with tauLacZ, X-gal staining signals were mostly localized in the layer II neurons in the occipital pole, being consistent with the in situ hybridization result. Fluorescent immunohistochemistry using anti-beta-galactosidase antibody further detected the tauLacZ signals in the same cells. X-gal staining began at P3, peaked at P8, and continued to adults, although signals gradually decreased. These data showed that at least a few odorant receptors are expressed not only in olfactory sensory neurons but also in pyramidal neurons in the cerebral cortex, possibly playing an important role either in chemical detection of exogenous or endogenous ligands or in a developmental process such as axon guidance and target recognition.     

Effects of Corn Steep Liquor and Thiamine on l-Glutamic Acid Fermentation of Hydrocarbons: IV. Utilization of Hydrocarbons by Microorganisms

The effect of nutrients of natural source, such as corn steep liquor, peptone, and yeast extract, on the fermentative production of L-glutamic acid from hydrocarbons by a Corynebacterium was studied. Corn steep liquor and meat extract were found to be remarkably stimulatory to L-glutamic acid production; about 5 g per liter of L-glutamic acid were accumulated in a culture broth containing 3% n-paraffins, 0.01% corn steep liquor, and mineral salts. Among nutritional factors contained in corn steep liquor, biotin had very little effect on the accumulation of L-glutamic acid, but thiamine was highly stimulatory to L-glutamic acid production. The optimal concentration of thiamine for L-glutamic acid production was 3 to 5 μg per liter, and for cell growth, 50 μg per liter. L-Glutamic acid was accumulated in negligible quantity when the amount of thiamine in the culture broth was sufficient to support abundant growth of bacterial cells.     

A Cre knock‐in mouse line on the Sickle tail locus induces recombination in the notochord and intervertebral disks

Sickle tail (Skt) was originally identified by gene trap mutagenesis in mice, and the trapped gene is highly expressed in the notochord, intervertebral discs (IVD), and mesonephros. Here, we report the generation of Skt^cre mice expressing Cre recombinase in the IVD due to target insertion of the cre gene into the Skt locus by recombinase‐mediated cassette exchange. Crossing a conditional lacZ Reporter (R26R), Cre expression from the Skt^cre allele specifically activates β‐galactosidase expression in the whole notochord from E9.5 onwards. In E15.5 Skt^cre;R26R embryos, reporter activity was detected in the nucleus pulposus and in a portion of the annulus fibrosus, resulting in expansion of Cre‐expressing cells in the adult IVD. Reporter activity was also seen in the Skt^cre;R26R mesonephros at E15.5. These results suggest that Skt^cre mice are useful for exploring the fate specification of notochordal cells and creating models for IVD‐related skeletal diseases. genesis 50:758–765, 2012. © 2012 Wiley Periodicals, Inc.     

Discrimination of the sibling species Apodemus flavicollis and A. sylvaticus (Rodentia,Muridae)

Karyotyping and several molecular methods have allowed successful identification of two morphologically similar wide-ranging Western Palearctic species, the yellow-necked field mouse Apodemus flavicollis (Melchior, 1934) and the long-tailed wood mouse A. sylvaticus (Linnaeus, 1758), but reliable species diagnosis on the basis of morphometric characters is particularly problematic. Although they are easily morphologically distinguishable in Central and Northern Europe, this is not the case in southern parts of their distribution areas. Despite that, we have successfully discriminated A. flavicollis and A. sylvaticus from Serbia (Southern Europe) using geometric and traditional morphometric methods on a data set for ventral crania of specimens previously genotyped by the Inter Simple Sequence Repeat-PCR (ISSR-PCR). Discrimination power of applied approaches was more or less similar. The majority of our results were consistent with those obtained for specimens collected across the Czech Republic (Central Europe). Morphological differences observed herein, as well as those already reported between A. flavicollis and A. sylvaticus from the central and northern parts of their distribution areas, could be the outcome of their biology, i.e. ecological discrepancies, different assumed evolutionary scenarios considering biogeography, phylogeny, history and ontogeny.     

11C]sorafenib: radiosynthesis and preliminary PET study of brain uptake in P-gp/Bcrp knockout mice

Sorafenib (Nexavar, BAY43-9006, 1) is a second-generation, orally active multikinase inhibitor that is approved for the treatment of some cancers in patients. In this Letter, we developed [¹¹C]1 as a novel positron emission tomography (PET) probe, and evaluated the influence of ABC transporters-mediated efflux on brain uptake using PET with [¹¹C]1 in P-glycoprotein (P-gp)/breast cancer resistance protein (Bcrp) knockout mice versus wild-type mice. [¹¹C]1 was synthesized by the reaction of hydrochloride of aniline 2 with [¹¹C]phosgene ([¹¹C]COCl₂) to give isocyanate [¹¹C]6, followed by reaction with another aniline 3. Small-animal PET study with [¹¹C]1 indicated that the radioactivity level (AUC_0-60 min, SUV × min) in the brains of P-gp/Bcrp knockout mice was about three times higher than in wild-type mice.     

Radiosynthesis and preliminary evaluation of 4-[18F]fluoro-N-[4-[6-(isopropylamino)pyrimidin-4-yl]-1,3-thiazol-2-yl]-N-methylbenzamide as a new positron emission tomography ligand for metabotropic glutamate receptor subtype 1

The purpose of this study was to develop 4-[¹⁸F]fluoro-N-[4-[6-(isopropylamino)pyrimidin-4-yl]-1,3-thiazol-2-yl]-N-methylbenzamide ([¹⁸F]FITM, [¹⁸F]4) as a new PET ligand for imaging metabotropic glutamate receptor subtype 1 (mGluR1). [¹⁸F]4 was synthesized by [¹⁸F]fluorination of a novel nitro precursor 3 with [¹⁸F]KF in the presence of Kryptofix 222. At the end of synthesis, 429-936 MBq (n = 8) of [¹⁸F]4 was obtained with >99% radiochemical purity and 204-559 GBq/μmol specific activity starting from 6.7 to 13.0 GBq of [¹⁸F]F⁻. The brain distribution of [¹⁸F]4 was determined by the in vitro and ex vivo autoradiography using rat brain sections. The in vitro and in vivo specific binding of [¹⁸F]4 to mGluR1 was detected in the cerebellum, thalamus, hippocampus, and striatum. These results suggest that [¹⁸F]4 is a promising PET ligand for the in vivo evaluation of mGluR1.     

Functional involvement of dual specificity phosphatase 16 (DUSP16), a c-Jun N-terminal kinase-specific phosphatase, in the regulation of T helper cell differentiation

Naïve CD4⁺ T helper (Th) cells differentiate into distinct subsets of effector cells (Th1, Th2, Th17, and induced regulatory T cells (iTreg)) expressing different sets of cytokines upon encounter with presented foreign antigens. It has been well established that Th1/Th2 balance is critical for the nature of the following immune responses. Previous reports have demonstrated important roles of c-Jun N-terminal kinase (JNK) in Th1/Th2 balance, whereas the regulatory mechanisms of JNK activity in Th cells have not been elucidated. Here, we show that dual specificity phosphatase 16 (DUSP16, also referred to as MKP-M or MKP-7), which preferentially inactivates JNK, is selectively expressed in Th2 cells. In the in vitro differentiation assay of naïve CD4⁺ cells, DUSP16 expression is up-regulated during Th2 differentiation and down-regulated during Th1 differentiation. Chromatin immunoprecipitation revealed the increased acetylation of histone H3/H4 at the dusp16 gene promoter in CD4⁺ T cells under the Th2 condition. Adenoviral transduction of naïve CD4⁺ T cells with DUSP16 resulted in increased mRNA expression of IL-4 and GATA-3 in Th2 and decreased expression of IFNγ and T-bet in Th1 differentiation. In contrast, transduction of a dominant negative form of DUSP16 had the reverse effects. Furthermore, upon immunization, T cell-specific dusp16 transgenic mice produced antigen-specific IgG2a at lower amounts, whereas DN dusp16 transgenic mice produced higher amounts of antigen-specific IgG2a accompanied by decreased amounts of antigen-specific IgG1 and IgE than those of control mice. Together, these data suggest the functional role of DUSP16 in Th1/Th2 balance.     

Impaired flow-dependent control of vascular tone and remodeling in P2X4-deficient mice

The structure and function of blood vessels adapt to environmental changes such as physical development and exercise. This phenomenon is based on the ability of the endothelial cells to sense and respond to blood flow; however, the underlying mechanisms remain unclear. Here we show that the ATP-gated P2X4 ion channel, expressed on endothelial cells and encoded by P2rx4 in mice, has a key role in the response of endothelial cells to changes in blood flow. P2rx4(-/-) mice do not have normal endothelial cell responses to flow, such as influx of Ca(2+) and subsequent production of the potent vasodilator nitric oxide (NO). Additionally, vessel dilation induced by acute increases in blood flow is markedly suppressed in P2rx4(-/-) mice. Furthermore, P2rx4(-/-) mice have higher blood pressure and excrete smaller amounts of NO products in their urine than do wild-type mice. Moreover, no adaptive vascular remodeling, that is, a decrease in vessel size in response to a chronic decrease in blood flow, was observed in P2rx4(-/-) mice. Thus, endothelial P2X4 channels are crucial to flow-sensitive mechanisms that regulate blood pressure and vascular remodeling.