Angiostatin production in cultivation of recombinant Pichia pastoris fed with mixed carbon sources

A recombinant strain of Pichia pastoris with a phenotype of Mut^S was used to produce angiostatin. Due to the low methanol consumption rate of this strain, both methanol and glycerol feedings, that produced oscillation in dissolved O₂ concentration, were used during the expression phase to improve cell growth and angiostatin expression. However, enhanced cell growth led to nitrogen limitation that suppressed further production of angiostatin, but addition of ammonia allowed angiostatin concentration to reach 108 mg l^–1 after an expression period of 96 h. The ratio of consumed glycerol to methanol of 1.5:1 (w/w) in the expression phase suggested that methanol played an important role in the metabolism of carbon sources.     

The structure-anticoagulant activity relationships of sulfated lacquer polysaccharide: effect of carboxyl group and position of sulfation

Regiospecific oxidation of the primary hydroxyl groups in lacquer polysaccharide (LPL, Mw 6.85 x 10(4)) and its NaIO4 oxidation derivatives (LPLde) to C-6 carboxy groups was achieved with NaOCl in the presence of Tempo and NaBr. Sulfate groups were incorporated into the oxidated polysaccharides using Py.SO3 complex as a reagent. Reactivity of polysaccharide hydroxyl group was C-6 > C-2 > C-4. Sulfate groups were mainly linked to the second hydroxy at C-2 in the products. The results of APTT assay showed after incorporation of carboxyl groups into lacquer polysaccharides, the intrinsic coagulation pathway was promoted, and all sulfated polysaccharides had very weak anticoagulant activity within the scope of studied DS (0.39-1.11). These indicated that carboxyl groups and sulfate groups had the synergistic action. At the same time, the anticoagulant activity increased very slowly with the DS in the second hydroxy. This indicated that 6-O-SO3- in the side chains took an important role in the anticoagulant activity.     

可溶性淀粉合成酶SSⅢ基因对马铃薯的遗传转化

马铃薯是淀粉生产中重要的农作物之一,而可溶性淀粉合成酶SSⅢ是可溶性淀粉合成酶的主要活性成分,通过基因工程的手段来研究SSⅢ基因在淀粉合成中的功能可以用于改良马铃薯淀粉的品质.本研究采用根癌农杆菌介导法将强组成型表达启动子CaMV 35S驱动的可溶性淀粉合成酶SSⅢ基因的RNA干扰表达载体导入马铃薯栽培品种克新1号和克新4号中,获得了65株卡那霉素抗性植株.对抗性植株PCR检测结果表明,SSⅢ基因的干扰片段已整合到马铃薯基因组中,RT-PCR检测表明SSⅢ基因在转录水平上受到了明显抑制.该研究为马铃薯淀粉品质的改良奠定了基础.     

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.     

Recent changes in body weight and wing length among some British passerine birds

We tested the prediction that global warming has caused recent decreases in body weight (Bergmann's rule) and increases in wing length (Allen's rule) in 14 species of passerine birds at two localities in England: Wicken Fen (1968–2003) and Treswell Wood (1973–2003).
Predicted long-term linear decreases in residual body weight occurred in four species: dunnocks (Wicken Fen), and great tits, blue tits and bullfinches (Treswell Wood). Non-linear decreases also occurred in reed warblers and blackcaps at Wicken Fen, which also had a surprising linear increase in residual body weight in blackbirds.
Residual wing lengths increased linearly, as predicted, in six of seven species at Wicken Fen. Whereas there were non-linear long-term increases in wrens, dunnocks and blackbirds in Treswell Wood. Unexpected linear decreases also occurred in residual wing lengths in willow warblers (Wicken Fen), and blue tits, great tits and chaffinches (Treswell Wood).
The most parsimonious explanation for such long-term changes in body weight is global warming, as predicted by Bergmann's rule. Greater site and species-specific effects on wing length (e.g. non-linear changes plus shorter wings in the woodland habitat) suggest a less straightforward conclusion concerning Allen's rule, probably because wing length involved variation in both bone and feather growth.
Changes in residual body weights and wing lengths often differed between species and were sometimes non-linear, perhaps reflecting short-term modifications in selection pressures. Human-induced influences are discussed, such as avian predator population densities and land-use change. Short-term variation in temperature had little effect, but rainfall did explain the unusual increase in blackbird body weight, possibly as a result of improving food (earthworm) availability.     

Coordination of endoplasmic reticulum and mRNA localization to the yeast bud

Localization of messenger RNAs and local protein synthesis contribute to asymmetric protein distribution not only of cytoplasmic but also of membrane or secreted proteins. Since synthesis of the latter protein classes occurs at the rough endoplasmic reticulum (ER), mRNA localization and distribution of ER should be coordinated. However, this coordination is not yet understood. In yeast, mRNA localization to the growing bud depends on the myosin Myo4p, its adaptor She3p, and the specific RNA binding protein She2p. These proteins mediate the localization of 23 mRNAs including ASH1 mRNA and mRNAs encoding membrane proteins. In addition, Myo4p and She3p are required for segregation of cortical ER to the bud. Here we show, with ASH1 mRNA as a model mRNA, that localizing messenger ribonucleoprotein (mRNP) particles comigrate with tubular ER structures to the bud, which requires the RNA binding protein She2p. Coordinated movement of the ASH1 mRNP with ER tubules but not their association with each other depends on Myo4p and She3p. Subcellular fractionation experiments demonstrate a cosegregation of ER and She2p, which is independent of Myo4p, She3p, or polysomes. Our findings suggest a novel model for mRNA localization that involves association of She2p and mRNPs with ER tubules and myosin-dependent cotransport of tubules and localized mRNPs.     

用彗星实验技术分析MTX对小鼠细胞DNA的损伤作用

MTX是一种抗叶酸药物 ,作用于增殖细胞 ,为了解其作用机制和探测其遗传毒性靶器官 ,以小鼠为研究对象 ,用彗星实验技术检测了MTX腹腔注射染毒后对脾、骨髓、胸腺、和外周血淋巴细胞的DNA损伤作用及其与MTX剂量间的相关。 1.2 5～ 5mg/kgMTX可诱发小鼠体内 4种细胞的DNA单链断裂 ,核DNA损伤程度与用药剂量呈正相关。不同种类细胞对MTX的易感性不同 ,脾、骨髓、胸腺、外周血淋巴细胞可能是MTX的遗传毒性靶细胞。外周血淋巴细胞在SCGE分析中的拖尾现象可作为用药后组织器官对药物敏感性反映的生物标志     

Endothelial adhesion molecule ESAM binds directly to the multidomain adaptor MAGI-1 and recruits it to cell contacts

Endothelial cell-selective adhesion molecule (ESAM) is an immunoglobulin-like transmembrane protein associated with endothelial tight junctions (TJ). Based on a yeast two-hybrid screen, we have identified the membrane-associated guanylate kinase protein MAGI-1 as an intracellular binding partner of ESAM. MAGI-1 is a multidomain adaptor protein, which binds to transmembrane, cytoskeletal, and signaling molecules, and has been localized to tight junctions in epithelial cells. MAGI-1 associates with the very C-terminal sequence of ESAM most likely through a PDZ domain-mediated interaction. The direct interaction between ESAM and MAGI-1 was confirmed by pull-down experiments. The two proteins formed stable complexes in transfected Chinese hamster ovary (CHO) cells, which could be immunoisolated. We found MAGI-1 to be associated with cell-cell contacts in human umbilical vein endothelial cells (HUVECs) and in mouse endothelium, where it colocalizes with ESAM. In CHO cells, recruitment of MAGI-1 to cell contacts required the presence of ESAM. Hence, ESAM may be involved in anchoring MAGI-1 at endothelial tight junctions.     

NMR structures of loop B RNAs from the stem-loop IV domain of the enterovirus internal ribosome entry site: a single C to U substitution drastically changes the shape and flexibility of RNA

The 5'-untranslated region of positive-strand RNA viruses harbors many cis-acting RNA structural elements that are important for various viral processes such as replication, translation, and packaging of new virions. Among these is loop B RNA of the stem-loop IV domain within the internal ribosomal entry site (IRES) of enteroviruses, including Poliovirus type 1 (PV1). Studies on PV1 have shown that specific recognition of loop B by the first KH (hnRNP K homology) domain of cellular poly(rC)-binding protein 2 (PCBP2) is essential for efficient translation of the viral mRNA. Here we report the NMR solution structures of two representative sequence variants of enteroviral loop B RNA. The two RNA variants differ at only one position (C vs U) within a six-nucleotide asymmetric internal loop sequence that is the binding site for the PCBP2 KH1 domain. Surprisingly, the two RNAs are drastically different in the overall shape and local dynamics of the bulge region. The RNA with the 5'-AUCCCU bulge sequence adopts an overall L shape. Its bulge nucleotides, especially the last four, are highly flexible and not very well defined by NMR. The RNA with the 5'-AUUCCU bulge sequence adopts an overall U shape, and its bulge sequence exhibits only limited flexibility. A detailed analysis of the two RNA structures and their dynamic properties, as well as available sequence data and known KH domain-RNA complex structures, not only provides insights into how loop B RNA might be recognized by the PCBP2 KH1 domain but also suggests a possible correlation between structural flexibility and pre-existing structural features for protein recognition.     

蛇毒类凝血酶鼠单克隆抗体的制备

采用杂交瘤技术,获得了４株稳定分泌抗蛇毒类凝血酶的单克隆抗体杂交瘤细胞株,均属ＩｇＧ１ｋ链,４株杂交瘤细胞培养上清液效价为 ４ × １０－１～４ × １０－２,腹水效价为 ４ × １０－１～３．２ ×１０－５。