Fringe glycosyltransferases differentially modulate Notch1 proteolysis induced by Delta1 and Jagged1

Fringe O-fucose-beta1,3-N-acetylglucosaminyltransferases modulate Notch signaling by potentiating signaling induced by Delta-like ligands, while inhibiting signaling induced by Serrate/Jagged1 ligands. Based on binding studies, the differential effects of Drosophila fringe (DFng) on Notch signaling are thought to result from alterations in Notch glycosylation that enhance binding of Delta to Notch but reduce Serrate binding. Here, we report that expression of mammalian fringe proteins (Lunatic [LFng], Manic [MFng], or Radical [RFng] Fringe) increased Delta1 binding and activation of Notch1 signaling in 293T and NIH 3T3 cells. Although Jagged1-induced signaling was suppressed by LFng and MFng, RFng enhanced signaling induced by either Delta1 or Jagged1, underscoring the diversity of mammalian fringe glycosyltransferases in regulating signaling downstream of different ligand-receptor combinations. Interestingly, suppression of Jagged1-induced Notch1 signaling did not correlate with changes in Jagged1 binding as found for Delta1. Our data support the idea that fringe glycosylation increases Delta1 binding to potentiate signaling, but we propose that although fringe glycosylation does not reduce Jagged1 binding to Notch1, the resultant ligand-receptor interactions do not effectively promote Notch1 proteolysis required for activation of downstream signaling events.     

Base selectivity is impaired by mutants that perturb hydrogen bonding networks in the RB69 DNA polymerase active site

To investigate the molecular basis for the selective utilization of nucleoside triphosphates complementary to templating bases, by RB69 DNA polymerase (RB69 pol), we constructed a set of mutants that we predicted would perturb the "floor" of the nascent base-pairing interface in the enzyme. We then determined the pre-steady-state kinetic parameters for the incorporation of complementary and noncomplementary dNTPs by the exo(-) form of RB69 pol and its mutants. We found that the Y567A mutant had the same K(d) and k(pol) values for incorporation of C versus G as the wild-type exo(-) enzyme; however, the k(pol)/K(d) ratio for G versus G incorporation with the Y567A mutant was 10 times higher than the k(pol)/K(d) efficiency of G versus G incorporation using the exo(-) RB69 pol. The reduced level of discrimination by the Y567A mutant against incorporation of mismatched bases was also seen with the Y391A mutant. Stopped-flow fluorescence was also employed to monitor rates of putative conformational changes with the exo(-) RB69 pol and its mutants using a primer-template complex containing 2-aminopurine. The rates of fluorescence changes were equal to or greater than the rates of the rapid chemical quench, indicating that we were monitoring a process occurring before or during the phosphoryl transfer reaction. We have interpreted our results within the context of the crystal structure of the RB69 pol ternary complex [Franklin, M. C., et al. (2001) Cell 105, 657-667].     

Aluminium resistance requires resistance to acid stress: a case study with spinach that exudes oxalate rapidly when exposed to Al stress

Spinach is a vegetable with a high oxalate concentration in its tissues. Oxalate efflux from spinach (Spinacia oleracea L. cv. Quanneng) roots was rapidly stimulated (within 30 min) by aluminium (Al) treatment. The efflux was constant within 6 h, but increased with increasing Al concentration. The efflux was confined to the root tip (0-5 mm), which showed a 5-fold greater efflux than the root zone distal to the tip (5-10 mm). Oxalate efflux could not be triggered by treatment with the trivalent cation lanthanum or by phosphorus deficiency, indicating that the efflux was specific to the Al treatment. All this evidence suggested that spinach possesses Al-resistance mechanisms. However, spinach was found to be as sensitive to Al toxicity as the Al-sensitive wheat line ES8, which had no Al-dependent organic acids efflux. The Al accumulated in the apical 5 mm of the roots of spinach which was also similar to that in the Al-sensitive wheat after 24 h treatment with 50 microM AlCl(3), indicating a non-exclusion mechanism. In addition, root elongation in spinach was significantly inhibited at pH 4.5, compared with that at pH 6.5. Based on this evidence, it is concluded that the sensitivity to acid stress in spinach could mask the potential role for oxalate to protect the plant roots from Al toxicity.     

Genes "waiting" for recruitment by the adaptive immune system: the insights from amphioxus

In seeking evidence of the existence of adaptive immune system (AIS) in ancient chordate, cDNA clones of six libraries from a protochordate, the Chinese amphioxus, were sequenced. Although the key molecules such as TCR, MHC, Ig, and RAG in AIS have not been identified from our database, we demonstrated in this study the extensive molecular evidence for the presence of genes homologous to many genes that are involved in AIS directly or indirectly, including some of which may represent the putative precursors of vertebrate AIS-related genes. The comparative analyses of these genes in different model organisms revealed the different fates of these genes during evolution. Their gene expression pattern suggested that the primitive digestive system is the pivotal place of the origin and evolution of the AIS. Our studies support the general statement that AIS appears after the jawless/jawed vertebrate split. However our study further reveals the fact that AIS is in its twilight in amphioxus and the evolution of the molecules in amphioxus are waiting for recruitment by the emergence of AIS.     

Loss of viability during freeze–thaw of intact and adherent human embryonic stem cells with conventional slow-cooling protocols is predominantly due to␣apoptosis rather than cellular necrosis

Summary A major challenge in the widespread application of human embryonic stem (hES) cells in clinical therapy and basic scientific research is the development of efficient cryopreservation protocols. Conventional slow-cooling protocols utilizing standard cryoprotectant concentrations i.e. 10% (v/v) DMSO, yield extremely low survival rates of <5% as reported by previous studies. This study characterized cell death within frozen–thawed hES colonies that were cryopreserved under standard conditions. Surprisingly, our results showed that immediately after post-thaw washing, the overwhelming majority of hES cells were viable (≈98%), as assessed by the trypan blue exclusion test. However, when the freshly-thawed hES colonies were incubated within a 37 °C incubator, there was observed to be a gradual reduction in cell viability over time. The kinetics of cell death was drastically slowed-down by keeping the freshly-thawed hES colonies at 4 °C, with >90% of cells remaining viable after 90 min of incubation at 4 °C. This effect was reversible upon re-exposing the cells to physiological temperature. The vast majority of low temperature-exposed hES colonies gradually underwent cell death upon incubation for a further 90 min at 37 °C. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end-labeling (TUNEL) assay confirmed apoptosis-induced nuclear DNA fragmentation in frozen–thawed hES cells after incubation at 37 °C for 90 min. Expression of active caspase-3 enzyme, which is another prominent marker of apoptosis, was confirmed by immunocytochemical staining, while transmission electron microscopy showed typical ultrastructural features of apoptosis such as chromatin condensation and margination to the nuclear membrane. Hence, our results demonstrated that apoptosis instead of cellular necrosis, is the major mechanism of the loss of viability of cryopreserved hES cells during freeze–thawing with conventional slow-cooling protocols.     

我国转基因Bt抗虫棉的进展分析与生态风险评估

从源头创新、基因转移技术、新品种选育与推广、专利分析四个方面探讨了我国转基因Bt抗虫棉的重大进展。且从靶标昆虫与非目标昆虫两个方面重点对转基因的生态风险进行了综合评价,并根据国内外的研究趋势,提出了加速我国转基因棉研究的对策。     

Role of nuclear chromogranin B in inositol 1,4,5-trisphosphate-mediated nuclear Ca2+ mobilization

Recently, secretory granule Ca(2+) storage protein chromogranin B (CGB) was shown to be present in the nucleoplasm proper in a complex structure that consists of the inositol 1,4,5-trisphosphate receptor (IP(3)R)/Ca(2+) channels and the phospholipids. Further, the amounts of IP(3)Rs present in the nucleus of bovine chromaffin cells were shown to be comparable to that of the endoplasmic reticulum. Therefore, we investigated here the potential contribution of nuclear CGB on the IP(3)-dependent Ca(2+) mobilization in the nucleus, using both neuroendocrine PC12 and nonneuroendocrine NIH3T3 cells. Chromogranin A (CGA) expression in the NIH3T3 cells, which do not contain intrinsic chromogranins, increased the IP(3)-induced Ca(2+) releases in the nucleus by 45%, while CGB expression in the same cells increased the IP(3)-induced Ca(2+) releases in the nucleus by 80%. Microinjection of IP(3) into the nucleus of CGB-expressing NIH3T3 cells increased the IP(3)-dependent nuclear Ca(2+) mobilization approximately 3-fold, whereas in CGA-expressing cells it remained the same as that of control cells. In contrast, inhibition of CGA expression in PC12 cells by siRNA treatment decreased the IP(3)-induced Ca(2+) releases in the nucleus by 17%, while inhibition of CGB expression decreased the IP(3)-induced Ca(2+) releases in the nucleus by 55%. Microinjection of IP(3) into the nucleus of siCGB-treated PC12 cells decreased the IP(3)-dependent nuclear Ca(2+) mobilization by approximately 75%, whereas in siCGA-treated cells it remained the same as that of control cells. Given the presence of CGB in the nucleus, these results further highlight the critical contribution of nuclear CGB in the IP(3)-induced Ca(2+) release in the nucleus.     

大鼠原代肝细胞培养方法的建立

目的：探索混合胶原凝胶培养肝细胞的方法,观察培养鼠肝细胞的功能与形态特征,用于评价中药十八反的作用机理。方法：两步法分离鼠肝细胞,与鼠尾胶原溶液混合接种于培养板,观察培养鼠肝细胞的形态学特征和生化指标。采用RT-PCR技术。检测药物对P450亚酶CYP3A1表达的影响,进一步确证该体系的可靠性。结果：双层胶原培养体系可观察到典型的肝细胞形态特征,肝细胞功能检测显示肝细胞合成分泌的尿素、白蛋白,而乳酸脱氢酶漏出量较少。药物对P450亚酶CYP3A1表达的影响呈良好的剂量依赖性,同时双层胶原具有保持肝细胞活性的优点。可作为原代肝细胞培养的条件。结论：混合胶原凝胶培养能保留体内的细胞功能和活性,特别是保留药物代谢酶的活性。     

Characterization and identification of Sox2+ radial glia cells derived from rat embryonic cerebral cortex

During the central nervous system (CNS) development, radial glia cells (RGCs) play at least two essential roles, they contribute to neuronal production and the subsequent guidance of neuronal migration, whereas its precise distribution and contribution to cerebral cortex remains less understood. In this research, we used Vimentin as an astroglial marker and Sox2 as a neural progenitor marker to identify and investigate RGCs in rat cerebral cortex at embryonic day (E) 16.5. We found that the Sox2+ progenitor cells localized in the germinal zone (GZ) of E16.5 cerebral cortex, ~95% Sox2+ cells co-localized with Vimentin+ or Nestin+ radial processes which extended to the pial surface across the cortical plate (CP). In vitro, we obtained RG-like cells from E16.5 cerebral cortex on adherent conditions, these Sox2+ Radial glia (RG)-like cells shared some properties with RGCs in vivo, and these Sox2+ RG-like cells could differentiate into astrocytes, oligodendrocytes and presented the radial glia—neuron lineage differentiation ability. Taken together, we identified and investigated some characterizations and properties of Sox2+ RGCs derived from E16.5 cerebral cortex, we suggested that the embryonic Sox2+ progenitor cells which located in the cortical GZ were mainly composed of Sox2+ RGCs, and the cortex-derived Sox2+ RG-like cells displayed the radial glia—neuron lineage differentiation ability as neuronal progenitors in vitro.