Regulatory BC1 RNA and the fragile X mental retardation protein: convergent functionality in brain

Background

BC RNAs and the fragile X mental retardation protein (FMRP) are translational repressors that have been implicated in the control of local protein synthesis at the synapse. Work with BC1 and Fmr1 animal models has revealed that phenotypical consequences resulting from the absence of either BC1 RNA or FMRP are remarkably similar. To establish functional interactions between BC1 RNA and FMRP is important for our understanding of how local protein synthesis regulates neuronal excitability.

Methodology/Principal Findings

We generated BC1−/− Fmr1−/− double knockout (dKO) mice. We examined such animals, lacking both BC1 RNA and FMRP, in comparison with single knockout (sKO) animals lacking either one repressor. Analysis of neural phenotypical output revealed that at least three attributes of brain functionality are subject to control by both BC1 RNA and FMRP: neuronal network excitability, epileptogenesis, and place learning. The severity of CA3 pyramidal cell hyperexcitability was significantly higher in BC1−/− Fmr1−/− dKO preparations than in the respective sKO preparations, as was seizure susceptibility of BC1−/− Fmr1−/− dKO animals in response to auditory stimulation. In place learning, BC1−/− Fmr1−/− dKO animals were severely impaired, in contrast to BC1−/− or Fmr1−/− sKO animals which exhibited only mild deficits.

Conclusions/Significance

Our data indicate that BC1 RNA and FMRP operate in sequential-independent fashion. They suggest that the molecular interplay between two translational repressors directly impacts brain functionality.     

二氮嗪对自发性高血压大鼠心功能及心肌ERK和JNK表达的影响

目的:探讨二氮嗪对离体自发性高血压大鼠心脏缺血/再灌注心功能及心肌组织ERK和JNK表达的影响及可能机制。方法:雄性自发性高血压大鼠取心行Langendorff灌流。实验分为5组(n=6/组):对照组(Con)在平衡后继续灌流40min,全心缺血25min,复灌30min。其余各组除全心缺血前处理不同外,余均同对照组。缺血预处理组(IP)2次给予5min缺血+10min复灌,二氮嗪预处理组(DP)给予2次含50μmol·L-1二氮嗪的K-H液10min后给不含二氮嗪的K-H液5min,5-HD、5-HD+DP组则在平衡后给予10min150μmol·L-1线粒体KATP阻断剂5-HD,余同Con及DP组。结果:IP组及DP组复灌末左室发展压、+dP/dtmax和-dP/dtmax的恢复率均高于Con组(P<0.01),但两组左室舒张末期压恢复率低于Con组(P<0.01);5-HD能拮抗二氮嗪引起的心功能指标的改善。复灌末IP、DP及5-HD+DP组ERK表达增加。IP组及DP组心肌的JNK表达低于Con组(P<0.05),5-HD+DP组JNK表达显著高于DP组。结论:二氮嗪预处理对离体自发性高血压大鼠心肌缺血/再灌注损伤有保护作用,此保护作用可能与ERK的表达增加及JNK表达减少有关。     

Biological Synthesis of Semiconductor Zinc Sulfide Nanoparticles by Immobilized Rhodobacter sphaeroides

A novel, clean biological transformation reaction by immobilized Rhodobacter sphaeroides has been developed for the synthesis of zinc sulfide (ZnS) nanoparticles with an average diameter of 8 nm. The nanoparticles were examined by X-ray diffraction, transmission electron microscopy, energy dispersive analyses of X-rays, UV–vis optical absorption and photoluminescence spectra. The average diameter of ZnS nanoparticles varied according to the culture time.     

The flexible interrelation between AOX respiratory pathway and photosynthesis in rice leaves.

Alternative respiratory pathway was investigated in rice seedlings grown under total darkness, light/dark cycle, or continuous light. The capacity of the alternative pathway was relatively higher in leaves that had longer light exposure. An analysis of rice AOX1 multigene family revealed that AOX1c, but not AOX1a and AOX1b, had a light-independent expression. The alternative oxidase (AOX) inhibitor, salicylhydroxamic acid (SHAM, 1mM), inhibited nearly 68% of the capacity of the alternative pathway in leaves grown under different light conditions. The plants grown under different light periods were treated with SHAM and then were exposed to illumination for 4h. The transition from dark to 4h of light stimulated the capacity of alternative pathway in etiolated rice seedlings and in those grown under light/dark cycle, whereas the capacity of the alternative pathway was constant in seedlings grown under continuous light with additional 4h of illumination. Etiolated leaves did not show any CO(2) fixation after 4h of illumination, and the increase in chlorophyll content was delayed by the SHAM pretreatment. When seedlings grown under light/dark cycle were moved from dark and exposed to 4h of light, increases in chlorophyll content and CO(2) fixation rate were reduced by SHAM. Although these parameters were stable in plants grown under continuous light, SHAM decreased CO(2) fixation rate but not the chlorophyll content. These results indicate that the role and regulation of AOX in light are determined by the developmental stage of plant photosynthetic apparatus.     

Molecular Evolution of the Primate Antiviral Restriction Factor Tetherin

Background

Tetherin is a recently identified antiviral restriction factor that restricts HIV-1 particle release in the absence of the HIV-1 viral protein U (Vpu). It is reminiscent of APOBEC3G and TRIM5a that also antagonize HIV. APOBEC3G and TRIM5a have been demonstrated to evolve under pervasive positive selection throughout primate evolution, supporting the red-queen hypothesis. Therefore, one naturally presumes that Tetherin also evolves under pervasive positive selection throughout primate evolution and supports the red-queen hypothesis. Here, we performed a detailed evolutionary analysis to address this presumption.

Methodology/Principal Findings

Results of non-synonymous and synonymous substitution rates reveal that Tetherin as a whole experiences neutral evolution rather than pervasive positive selection throughout primate evolution, as well as in non-primate mammal evolution. Sliding-window analyses show that the regions of the primate Tetherin that interact with viral proteins are under positive selection or relaxed purifying selection. In particular, the sites identified under positive selection generally focus on these regions, indicating that the main selective pressure acting on the primate Tetherin comes from virus infection. The branch-site model detected positive selection acting on the ancestral branch of the New World Monkey lineage, suggesting an episodic adaptive evolution. The positive selection was also found in duplicated Tetherins in ruminants. Moreover, there is no bias in the alterations of amino acids in the evolution of the primate Tetherin, implying that the primate Tetherin may retain broad spectrum of antiviral activity by maintaining structure stability.

Conclusions/Significance

These results conclude that the molecular evolution of Tetherin may be attributed to the host–virus arms race, supporting the Red Queen hypothesis, and Tetherin may be in an intermediate stage in transition from neutral to pervasive adaptive evolution.     

Association of EXT1 and EXT2, hereditary multiple exostoses gene products, in Golgi apparatus

We prepared the specific antibodies for EXT1 and EXT2, hereditary multiple exostoses (HME) gene products, and characterized their expression, subcellular localization, and protein association among EXT members. Biochemical analyses indicate that EXT1 and EXT2 can associate and form homo/hetero-oligomers in vivo with or without HME-linked mutations, EXT1 (R340C) and EXT2 (D227N), when exogenously expressed in COS-7 cells. An immunocytochemical analysis showed that both EXT1 and EXT2 localized in Golgi apparatus, irrespective of HME mutations. An immunohistochemical analysis on developing bones further showed that both EXT1 and EXT2 were concomitantly expressed in hypertrophic chondrocytes of forelimb bones from 1-day-old neonatal mouse, but down-regulated in maturing chondrocytes of developing cartilage from 21-day-old mouse. Taken together with the recent finding that EXTs encode for the glycosyltransferase required for the synthesis of heparan sulfate [Lind, T., Tufaro, F., McCormick, C., Lindahl, U., and Lindholt, K. (1998) J. Biol. Chem. 273, 26265-26268], our results implied a molecular basis that a HME-linked mutation found in EXT genes could interfere the physiological function(s) of EXT homo/hetero-oligomers as glycosyltransferases in the developing bones of HME patients.     

Transesterification catalyzed by polystyrene-supported chymotrypsin in toluene: the effect of neutralization of basic or acidic groups attaching to polystyrene resins

Crosslinked polystyrene resins containing a low level of either basic or acidic groups were used for supports of alpha-chymotrypsin (CT), which catalyzed the transesterification of N-acetyl-L-phenylalanine ethyl ester (AcPheOEt) with propanol in toluene. With a minimal amount of water, CT was sorbed to the resins, basic or acidic groups of which were partly or fully neutralized by several soluble acids or bases. With an increasing degree of neutralization of basic resins by free acids, the rate of disappearance of AcPheOEt was decreased, whereas the by-product formation of AcPheOH, due to hydrolysis, was considerably suppressed, compared with the ester-exchange product, AcPheOPr. The pK(a) value of the neutralizing acid was also important for both CT activity and reaction selectivity. AcPheOPr was selectively produced at a certain range of pK(a) values. On the other hand, the neutralization of acidic resins with free amines enhanced the CT activity but a strong base promoted the formation of hydrolysis product. (c) 1995 John Wiley & Sons, Inc.     

One-step knockin for inducible expression in mouse embryonic stem cells

Transgenesis enables the elucidation of gene function; however, constant transgene expression is not always desired. The tetracycline responsive system was devised to turn on and off transgene expression at will. It has two components: a doxycycline (dox)-controlled transactivator (TA) and an inducible expression cassette. Integration of these transgenes requires two transfection steps usually accomplished by sequential random integration. Unfortunately, random integration can be problematic due to chromatin position effects, integration of variable transgene units, and mutation at the integration site. Therefore, targeted transgenesis and knockin were developed to target the TA and the inducible expression cassette to a specific location, but these approaches can be costly in time, labor, and money. Here, we describe a one-step Cre-mediated knockin system in mouse embryonic stem cells that positions the TA and inducible expression cassette to a single location. Using this system, we show dox-dependent regulation of eGFP at the DNA topoisomerase 3β promoter. Because Cre-mediated recombination is used in lieu of gene targeting, this system is fast and efficient.