Receptor proteins for photoreception have been studied for several decades. More recently, putative receptors for olfaction have been isolated and characterized. In contrast, no receptors for taste have been identified yet by molecular cloning. This report describes experiments aimed at identifying a receptor responsible for the taste of monosodium glutamate (MSG). Using reverse transcriptase (RT)-PCR, we found that several ionotropic glutamate receptors are present in rat lingual tissues. However, these receptors also could be detected in lingual tissue devoid of taste buds. On the other hand, RT-PCR and RNase protection assays indicated that a G-protein-coupled metabotropic glutamate receptor, mGluR4, also is expressed in lingual tissues and is limited only to taste buds. In situ hybridization demonstrated that mGluR4 is detectable in 40-70% of vallate and foliate taste buds but not in surrounding nonsensory epithelium, confirming the localization of this metabotropic receptor to gustatory cells. Expression of mGluR4 in taste buds is higher in preweaning rats compared with adult rats. This may correspond to the known higher sensitivity to the taste of MSG in juvenile rodents. Finally, behavioral studies have indicated that MSG and L-2-amino-4-phosphonobutyrate (L-AP4), a ligand for mGluR4, elicit similar tastes in rats. We conclude that mGluR4 may be a chemosensory receptor responsible, in part, for the taste of MSG.

为了构建α-synuclein-pEGFP真核表达载体，检测其在SH-SY5Y细胞内的表达，本研究应用下述方法：PCR扩增α- synuclein基因并消除终止密码子；PCR产物连入pGEM T-easy载体，经测序确认无误后，亚克隆入pEGFP-N1，构建α-synucle-in-pEGFP真核表达载体；LipofectAMINE法转染SH-SY5Y细胞；荧光显微镜检测报告基因表达产物EGFP，原位杂交和免疫荧光细胞化学法检测α-synucleinmRNA及其蛋白表达。结果显示，该载体转染SH-SY5Y细胞后，可在细胞内观察到报告基因和目的基因的表达产物。结论：α-synuclein-pEGFP真核表达载体构建成功，并可在细胞内表达。本工作为今后动态观察研究α-synu-clein致Parkinson病多巴胺能神经细胞损伤机制奠定基础。

We have shown previously that in vitro ischemia could induce apoptosis in primary culture of astrocytes. In this paper we demonstrate that astrocytes in culture could undergo apoptosis during in vitro incubation postischemia. We also measured the changes of phosphorylated Erk1/2 (p-Erk1/2) and phosphorylated Akt (p-Akt) in order to determine whether these two pathways play a role in apoptosis. After 4 h in vitro ischemic incubation of cultured astrocytes, a limited amount of nuclear condensation was demonstrated by Hoechst 33342 staining. When ischemic incubation was halted and the cultures transferred to standard normoxic incubation (postischemia) conditions, DNA fragmentation and apoptosis were demonstrated by TUNEL and DNA laddering analysis. TUNEL-positive astrocytes began to appear at 6 h postischemia and increased in number from 12 h postischemia. Western blot analysis showed that both p-Erk1/2 and p-Akt were elevated in astrocytes subjected to 4 h of ischemia. Elevated p-Erk1/2 levels were sustained during the postischemia incubation for 12 h and decreased significantly afterward, but did not return to the levels in the control cultures that did not experience ischemic insult. In contrast, the p-Akt level continued to increase at 6 and 12 h postischemia before declining significantly. The changes in p-Erk1/2 and p-Akt correlated well with the appearance of apoptotic astrocytes in the culture.

Substantial evidence has shown that extracellular signal-regulated kinases 1 and 2 (Erk1/2) and serine/threonine kinase (Akt) play important roles in regulating cell survival. We examined the activities of these kinases in astrocytes under ischemia in an anaerobic chamber. The level of phosphorylated Erk1/2 in astrocytes began to increase after 1 h ischemia, reached a maximum after 4 h ischemia, before decreasing from 5 to 6 h. Akt was activated later than Erk1/2. It was significantly increased after 4 h ischemia before declining steadily afterwards. Lactate dehydrogenase (LDH) assay and Hoechst nucleic staining indicated that U0126, which inhibits Erk1/2 phosphorylation, enhanced ischemia-induced cell death, whereas LY294002, which inhibits Akt phosphorylation, delayed cell death. These effects were dose-dependent. At 4 and 6 h ischemia, U0126-treated astrocytes expressed a lower level of Bcl-2 than controls. In contrast, LY294002-treated astrocytes expressed a higher level of Bcl-2 than controls as shown by Western blots. Bcl-xL expression level was not affected by either treatment. These data suggest that activation of the MAPK/Erk1/2 pathway might protect astrocytes from ischemic injury, but activation of the PI3-K/Akt pathway does not. The effect may involve Bcl-2 but not Bcl-xL expression.

In situ hybridization (ISH) using nonradioactive probes enables mRNAs to be detected with improved cell resolution but compromised sensitivity compared to ISH with radiolabeled probes. To detect rare mRNAs, we optimized several parameters for ISH using digoxygenin (DIG)-labeled probes, and adapted tyramide signal amplification (TSA) in combination with alkaline phosphatase (AP)-based visualization. This method, which we term TSA-AP, achieves the high sensitivity normally associated with radioactive probes but with the cell resolution of chromogenic ISH. Unlike published protocols, long RNA probes (up to 2.61KB) readily permeated cryosections and yielded stronger hybridization signals than hydrolyzed probes of equivalent complexity. RNase digestion after hybridization was unnecessary and led to a substantial loss of signal intensity without significantly reducing nonspecific background. Probe concentration was also a key parameter for improving signal-to-noise ratio in ISH. Using these optimized methods on rat taste tissue, we detected mRNA for mGluR4, a receptor, and transducin, a G-protein, both of which are expressed at very low abundance and are believed to be involved in chemosensory transduction. Because the effect of the tested parameters was similar for ISH on sections of brain and tongue, we believe that these methodological improvements for detecting rare mRNAs may be broadly applicable to other tissues.