Identification of a surface charged residue in the S3-S4 linker of the pacemaker (HCN) channel that influences activation gating

I(f), encoded by the hyperpolarization-activated cyclic nucleotide-modulated (HCN) channel family, is a key player in cardiac and neuronal pacing. Although HCN channels structurally resemble voltage-gated K(+) (Kv) channels, their structure-function correlation is much less clear. Here we probed the functional importance of the HCN1 S3-S4 linker by multiple substitutions of its residues. Neutralizing Glu(235), an acidic S3-S4 linker residue conserved in all hyperpolarization-activated channels, by Ala substitution produced a depolarizing activation shift (V(12) = -65.0 +/- 0.7 versus -70.6 +/- 0.7 mV for wild-type HCN1); the charge-reversed mutation E235R shifted activation even more positively (-56.2 +/- 0.5 mV). Increasing external Mg(2+) mimicked the progressive rightward shifts of E235A and E235R by gradually shifting activation (V(12) = 1 < 3 < 10 < 30 mm); Delta V(12) induced by 30 mm Mg(2+) was significantly attenuated for E235A (+7.9 +/- 1.2 versus +11.3 +/- 0.9 mV for wild-type HCN1) and E235R (+3.3 +/- 1.4 mV) channels, as if surface charges were already shielded. Consistent with an electrostatic role, the energetic changes associated with Delta V(12) resulting from various Glu(235) substitutions (i.e. Asp, Ala, Pro, His, Lys, and Arg) displayed a strong correlation with their charges (Delta Delta G = -2.1 +/- 0.3 kcal/mol/charge; r = 0.94). In contrast, D233E, D233A, D233G, and D233R did not alter activation gating. D233C (in C318S background) was also not externally accessible when probed with methanethiosulfonate ethylammonium (MTSEA). We conclude that the S3-S4 linker residue Glu(235) influences activation gating, probably by acting as a surface charge.     

Lysosomal enzyme oligosaccharide phosphorylation in mouse lymphoma cells: specificity and kinetics of binding to the mannose 6-phosphate receptor in vivo

Phosphomannosyl residues on lysosomal enzymes serve as an essential component of the recognition marker necessary for binding to the mannose 6-phosphate (Man 6-P) receptor and translocation to lysosomes. The high mannose-type oligosaccharide units of lysosomal enzymes are phosphorylated by the following mechanism: N-acetylglucosamine 1-phosphate is transferred to the 6 position of a mannose residue to form a phosphodiester; then N- acetylglucosamine is removed to expose a phosphomonoester. We examined the kinetics of this phosphorylation pathway in the murine lymphoma BW5147.3 cell line to determine the state of oligosaccharide phosphorylation at the time the newly synthesized lysosomal enzymes bind to the receptor. Cells were labeled with [2-(3)H]mannose for 20 min and then chased for various times up to 4 h. The binding of newly synthesized glycoproteins to the Man 6-P receptor was followed by eluting the bound ligand with Man 6-P. Receptor-bound material was first detected at 30 min of chase and reached a maximum at 60 min of chase, at which time approximately 10 percent of the total phosphorylated oligosaccharides were associated with the receptor. During longer chase times, the total quantity of cellular phosphorylated oligosaccharides decreased with a half-time of 1.4 h, suggesting that the lysosomal enzymes had reached their destination and had been dephosphorylated. The structures of the phosphorylated aligosaccharides of the eluted ligand were then determined and compared with the phosphorylated oligosaccharides of molecules which were not bond to the receptor. The major phosphorylated oligosaccharide species present in the nonreceptor-bound material contained a single phosphosphodiester at all time examined. In contrast, receptor-bound oligosaccharides were greatly enriched in species possessing one and two phosphomonoesters. These results indicate that binding of newly synthesized lysosomal enzymes to the Man 6-P receptor occurs only after removal of the covering N- acetylglucosamine residues.     

Trophic changes,without changes in the external nutrient loading

The impact of the fish population on trophic properties of lake water, was experimentally studied in an oligotrophic Swedish forest lake. Biotic changes following fish removal resulted in a development in oligotrophic direction as shown by the drop in limnetic primary production, pH, total phosphorus, total nitrogen and the increased transparency.     

玉米叶片生长部位渗透调节和生长的生物物理参数变化

玉米叶片生长部位随着水分胁迫加剧ψ_w降低、LER减慢。LER从最大到零,快速干旱处理的ψw从-0.55降至-0.85 MPa;缓慢干旱处理ψ_w从-0.88降至-1.13 MPa。在任何一种LER下,缓慢干旱处理的ψ_s比快速干旱处理更低,生长停止时,前者为-1.57 MPa,而后者为-1.30MPa。缓慢干旱叶片尽管在更低ψ_w下,仍能维持一定膨压,保持一定的生长速率。经历长时间水分胁迫会改变细胞延伸生长的生物物理参数,增大临界膨压(0.08～0.09 MPa)。这是水分胁迫植株,在一定ψ_p下生长速率减慢的原因。     

Alarm substance in Gyrinus aeratus (Coleoptera,Gyrinidae)

Gyrinid beetles are common in ponds and lakes in Sweden, where they aggregate in open areas of the surface. Gyrinid beetles have pygidial glands which produce compounds rendering them unpalatable to fish. This study examines whether the pygidial secretion can be used for alarm purposes in addition to other functions. Experiments showed that gyrinid beetles responded to water prepared with the pygidial compounds by evasive behaviour. The beetles did not respond when the compound was mediated by air. One component of the defense system of gyrinids is how they advertise their presence. The aggregation of dark beetles with their typical motion pattern when alerted is very conspicuous and easily identified by an experienced predator. We suggest that this aspect of gyrinid defence can be characterized as aposematic.     

Genetic Diversity of Global Aromatic Rice Varieties

Genetic diversity of 434 rice accessions collected from 16 countries, including 345 fragrance rice varieties and 89 non fragrance rice varieties, have been analyzed. A total of 573 alleles were detected by using 77 simple sequence repeats (SSR) primer pairs covering all rice 12 chromosomes. The value of allelic polymorphism information content (PIC) ranged from 0090 to 0845, with an average of 0516 per locus; Gene diversity (GD) varied from 0091 to 0859, with an average of 0573; The mean value of major allele frequencies (MAF) was 0540, covering from 0251 to 0953. In addition, 434 rice accessions are divided into three sub populations by cluster and population structure analysis, and FST between sub populations showed a mean of -0116, ranging from -0623 to 0494; The score of genetic distance calculated by Nei′s method appeared from 0207 to 0355. Major allele frequencies within each sub population distributed from 0408 to 0746, and gene diversity level from 0354 to 0699, while PIC from 0320 to 0658. Sequencing 6 mitochondrion genes in 18 rice varieties exhibited no different in 5 genes, whereas Mit4 contains a 3 SNPs in the gene body, which acts as an important marker to understanding the relationship between Indica/Japonica differentiation and the evolution of fragrant gene. Finally, genetic diversity and mitochondrion gene sequencing would help to know about the origin of fragrant resource and benefit rice breeding.     

妊娠对于父源性皮肤移植存活时间的影响

母胎耐受机制的阐明,将为器官移植免疫耐受方案的研究提供重要启示.本研究旨在阐明妊娠状态对父系来源移植皮片的存活是否有保护作用.2月龄雌性C57BL/6小鼠和2～4月龄BALB/c雄性小鼠同笼受孕.采用流式细胞技术确定妊娠过程中调节性T细胞（Treg）比例的时间变化规律.以单向混合淋巴细胞反应（MLR）手段比较研究妊娠对于父系来源脾细胞刺激后产生的增殖反应的影响.通过同种异体小鼠全厚皮片移植模型,观察妊娠对于父系来源移植皮片的存活是否具有保护作用.并用分子生物学技术研究此种效能的可能机制.结果显示,C57BL/6小鼠妊娠过程中,Treg占CD4＋T细胞的比例从妊娠前的4.2%逐渐上升,受孕8天左右达到高峰值（6.8%）,此后开始下降并逐渐回复至基线水平.MLR结果表明,针对父系来源脾细胞的刺激,妊娠组较对照组呈现显著的低反应性,其平均刺激指数分别是7.8和13.6（P〈0.05）.定量PCR研究表明,血红素加氧酶-1和吲哚胺2,3双加氧酶mRNA在胎盘高表达,在脾脏低表达（P〈0.05）.父系来源的移植皮片的平均存活时间在妊娠组和非妊娠组分别是7.67和7.08天,无统计学差异（P〉0.05）.由此认为,在小鼠妊娠过程中,尽管出现了具有免疫抑制功能的Treg的比例增加,尽管有针对父系来源刺激细胞的较低的MLR反应性,但是单次妊娠对于父系来源的移植皮片的存活,在本研究条件下,未能显示具有统计学意义的保护作用.     

利用重组自交系群体对黄瓜侧枝相关性状进行QTL定位分析

侧枝与黄瓜(Cucumis sativus)产量有密切关系. 本实验利用侧枝长势较弱、萌发较早的华北类型品系S94和侧枝长势较强、萌发较晚的欧洲类型品系S06构建的224个F6:7家系进行黄瓜侧枝相关性状的研究. 利用已构建的重组自交系群体遗传图谱, 使用软件WinQTLCart 2.5进行复合区间定位. 在2006年秋和2007年春两季, 共检测到4个侧枝相关性状(侧枝均长LBAL、侧枝总长LBTL、侧枝数目LBN和第一侧枝节位FLBN)的36个QTL, 单个QTL的贡献率在3.1%(lbtl2.1, 春季)~32.3%(lbn2.3, 春季)之间. 结果显示, 4个不同性状的11个QTL (lbal1.1, lbtl1.1, lbn1.2, flbn1.2等)在两季中都聚集在第1连锁群e23m18d~ME23EM6c之间(7.4 cM), 并且在第2连锁群的S94A1~ME4SA4a之间(13.9 cM)也检测到了4个不同性状的15个QTL (lbal2.1, lbtl2.1, lbn2.1和flbn2.1等). 两季共有21个QTL贡献率超过10%, 其中lbn2.3的贡献率(春季32.3%, LOD=18.4)为最大, lbtl1.3(秋季26.2%, LOD=17.4; 春季26.9%, LOD=17.9)在两季的位置和贡献率都稳定. 这些基因座为将来进行QTL精细定位提供了参考, 同时利用其紧密连锁(<10 cM)的特异标记(CMBR40, F, CS30, S94A1, CSWTA11B)可进行黄瓜侧枝性状的分子标记辅助育种.