Noggin and bFGF cooperate to maintain the pluripotency of human embryonic stem cells in the absence of feeder layers

Human embryonic stem (hES) cells are typically maintained on mouse embryonic fibroblast (MEF) feeders or with MEF-conditioned medium. However, these xenosupport systems greatly limit the therapeutic applications of hES cells because of the risk of cross-transfer of animal pathogens. Here we showed that the bone morphogenetic protein antagonist noggin is critical in preventing differentiation of hES cells in culture. Furthermore, we found that the combination of noggin and basic fibroblast growth factor (bFGF) was sufficient to maintain the prolonged growth of hES cells while retaining all hES cell features. Since both noggin and bFGF are expressed in MEF, our findings suggest that they may be important factors secreted by MEF for maintaining undifferentiated pluripotent hES cells. Our data provide new insight into the mechanism how hES cell self-renewal is regulated. The newly developed feeder-free culture system will provide a more reliable alternative for future therapeutic applications of hES cells.     

免疫刺激复合物疫苗制备及其对小鼠免疫功能的影响

确定了免疫刺激复合物(ISCOM)疫苗的安全有效剂量及其对小鼠免疫功能的影响。选取体重25 g左右的昆明小白鼠60只,分为12组,每组5只,腹腔分别注射不同剂量(5-200μg)的ISCOM疫苗,观察小鼠健康状态。选取体重28 g左右的昆明小白鼠60只,分为4组,分别在腹腔注射相同剂量的灭菌生理盐水,Lipase+生理盐水,空ISCOM,Lipase+ISCOM疫苗,利用间接ELISA检测血清中特异性抗体效价。结果表明,当注射剂量为5-25μg/只时,小鼠无任何异常症状。免疫后8 d,小鼠血清特异性抗体效价(log2)可以达到10.5,显著高于对照组。因此,ISCOM疫苗能有效引起小鼠的免疫反应。     

Enhancement of hyperthermia-induced apoptosis by local anesthetics on human histiocytic lymphoma U937 cells

The combined effects of hyperthermia at 44 degrees C and local anesthetics on apoptosis in human histiocytic lymphoma U937 cells were investigated. When the cells were exposed to hyperthermia for l0 min marginal DNA fragmentation and nuclear fragmentation were observed. In the presence of amide-type local anesthetics further enhancement was found depending on concentration. The order of the concentration required for maximum induction was the reverse order of the lipophilicity (prilocaine > lidocaine > bupivacaine). Western blotting revealed that in hyperthermia there was initial release of Ca(2+) from the intracellular store site as indicated by increased expression of the type 1 inositol-1,4,5-trisphosphate receptor. However, the combination with lidocaine did not induce any further enhancement. Lidocaine enhanced the decrease in ATP content and the increase in intracellular Ca(2+) concentration in individual cells induced by hyperthermia. In addition, superoxide formation, decrease in the mitochondrial membrane potential, and activation of intracellular caspase-3 were found in the cells treated with hyperthermia and lidocaine. All of these were suppressed in part in the presence of the intracellular Ca(2+) ion chelator BAPTA-AM (bis-(O-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl). The present results indicate that local anesthetics at optimal concentrations enhance hyperthermia-induced apoptosis via Ca(2+)- and mitochondria-dependent pathways. Initial release of Ca(2+) from intracellular store sites caused by hyperthermia and followed by the subsequent increase in the intracellular Ca(2+) concentration and the additional activation of the mitochondrial caspase-dependent pathway (partly regulated by intracellular Ca(2+) concentration) plays a crucial role in the enhancement of apoptosis induced by the combination of hyperthermia and lidocaine.     

Immobilization of oligodeoxyribonucleotides with multiple anchors to microchips

Facile modification of oligodeoxyribonucleotides is required for efficient immobilization to a pre-activated glass surface. This report presents an oligodeoxyribonucleotide which contains a hairpin stem–loop structure with multiple phosphorothioate moieties in the loop. These moieties are used to anchor the oligo to glass slides that are pre-activated with bromoacetamidopropylsilane. The efficiency of the attachment reaction was improved by increasing the number of phosphorothioates in the loop, as shown in the remarkable enhancement of template hybridization and single base extension through catalysis by DNA polymerase. The loop and stem presumably serve as lateral spacers between neighboring oligodeoxyribonucleotides and as a linker arm between the glass surface and the single-stranded sequence of interest. The oligodeoxyribonucleotides of this hairpin stem–loop architecture with multiple phosphorothioate moieties have broad application in DNA chip-based gene analysis.     

凉水保护区松鼠冬季重取食物的贮藏点与越冬生存策略

为了解松鼠贮食行为中的重取食物行为特征,以及在冬季不同生存环境条件下,重取贮藏的红松种子的状况,于2004年初冬(11月中旬),仲冬(2005年1月下旬)和晚冬(3月下旬)采用随机抽样法对凉水自然保护区内雪地上的松鼠重取食物后遗留的贮藏点(称为重取贮点)进行了调查和分析。结果表明:1)冬季不同时期松鼠的重取贮点强度存在明显差异,初冬最大,晚冬次之,仲冬最小,3 个时期的样本数据频率分布检验表明其总体皆服从指数分布;2)当场取食的贮藏点占到83%,17% 的贮藏点未发现籽壳遗留。受松籽低产量及人为过量收获的影响,单个贮藏点贮藏1 粒和2 粒种子的占92.9% ;3)受气温和雪被厚度因素的影响,3 个时期重取贮藏点的口径与深度存在显著差异,一定深度的雪被可能有助于松鼠的重取,但过深的雪被也许会使松鼠无法重取。本研究表明,本地区松鼠在冬季不同时期重取红松种子的强度分配符合将饥饿风险降至最小的觅食经济学原则;松鼠重取行为的优化在贮藏点的被取食状况、口径与深度等特征上得到体现。     

KPC1 expression and essential role after acute spinal cord injury in adult rat

KPC1 (Kip1 ubiquitylation-promoting complex 1) is the catalytic subunit of the ubiquitin ligase KPC, which regulates the degradation of the cyclin-dependent kinase inhibitor p27^kip1 at the G1 phase of the cell cycle. To elucidate the expression and role of KPC1 in nervous system lesion and repair, we performed an acute spinal cord contusion injury (SCI) model in adult rats. Western blot analysis showed a significant up-regulation of KPC1 and a concomitant down-regulation of p27^kip1 following spinal injury. Immunohistochemistry and immunofluorescence revealed wide expression of KPC1 in the spinal cord, including expression in neurons and astrocytes. After injury, KPC1 expression was increased predominantly in astrocytes, which highly expressed PCNA, a marker for proliferating cells. Co-immunoprecipitation demonstrated increased interactions between p27^kip1 and KPC1 4 days after injury. To understand whether KPC1 plays a role in astrocyte proliferation, we applied LPS to induce astrocyte proliferation in vitro. Western blot analysis demonstrated that p27^kip1 expression was negatively correlated with KPC1 expression following LPS stimulation. Immunofluorescence analysis showed subcellular localizations of p27^kip1 and KPC1 were also changed following the stimulation of astrocytes with LPS. These results suggest that KPC1 is related to the down-regulation of p27^kip1; this event may be involved in the proliferation of astrocytes after SCI.     

Determination of genetic relationships among five indigenous Chinese goat breeds with six microsatellite markers

Microsatellite variation was analyzed in five Chinese indigenous goat breeds, which include four Cashmere breeds (Tibetan, Neimonggol, Liaoning, Taihang) and one Hubei local breed (Matou) used for meat production. Five ovine and one bovine microsatellites, selected from eight ovine microsatellites and five bovine microsatellites were suitable for use in this study. With these six microsatellites, allele frequencies, heterozygosity, polymorphism information content (PIC) and effective allele number were calculated. A neighbor-joining tree was constructed using Nei's standard genetic distance (1978). In the tree, Neimonggol and Liaoning were grouped together, then with Taihang; while Tibetan and Matou individually had their own branch. The genetic relationship of five breeds corresponds to their history and geographic origins.     

Phycobilisome from Anabaena Variabilis Kütz. and its Model Conjugates

The model conjugates phycocyanin-allophycocyanin (C-PC-APC) and phycoerythrocyanin-phycocyanin-allophycocyanin (PEC-C-PC-APC) were synthesized by using a heterobifunctional coupling reagent N-succinimidyl-3-(2-pyridyldithio)propionate. The rod-core complex (αβ)₆ ^PCL_RC ²⁷(αβ)₃ ^APCL_C ^8.9 and phycobilisomes were separated from Anabaena variabilis. Energy transfer features for the conjugates and the complexes were compared. The absorption and fluorescence emission spectra indicated that the linker-peptides mediate interaction of phycobiliproteins and prompt energy transfer. The energy transfer in the conjugates was detected by fluorescence emission spectra and confirmed by the addition of dithiothreitol. The conjugates may be used as models for studying the energy transfer mechanism in phycobilisomes. This revised version was published online in September 2006 with corrections to the Cover Date.