Sweet potato (Ipomoea batatas) trypsin inhibitors expressed in transgenic tobacco plants confer resistance against Spodoptera litura

A sweet potato (Ipomoea batatas cv. Tainong 57) trypsin inhibitor gene was introduced into tobacco plants (Nicotiana tabaccum cv. W38) by Agrobacterium tumefaciens– mediated transformation. From 30 independent transformants, three lines with high level of expression were further analyzed. The trypsin inhibitor gene, under control of the 35S CaMV promoter, led to the production of the trypsin inhibitor proteins up to 0.2% of the total protein. In insecticidal bioassays of transgenic tobacco plants, larval, growth of Spodoptera litura (F.), the tobacco cutworm, was severely retarded as compared to their growth on control plants. This observation implied that expression of sweet potato trypsin inhibitor can provide an efficient method for crop protection. Received: 29 July 1996 / Revision received: 15 November 1996 / Accepted: 8 December 1996     

Molecular cloning,bioinformatics analysis and functional characterization of HWTX-XI toxin superfamily from the spider Ornithoctonus huwena

Spider venom contains a very valuable repertoire of natural resources to discover novel components for molecular diversity analyses and therapeutic applications. In this study, HWTX-XI toxins from the spider venom glands of Ornithoctonus huwena which are Kunitz-type toxins (KTTs) and were directly cloned, analyzed and functionally characterized. To date, the HWTX-XI superfamily consists of 38 members deduced from 121 high-quality expressed sequence tags, which is the largest spider KTT superfamily with significant molecular diversity mainly resulted from cDNA tandem repeats as well as focal hypermutation. Among them, HW11c40 and HW11c50 may be intermediate variants between native Kunitz toxins and sub-Kunitz toxins based on evolutionary analyses. In order to elucidate their biological activities, recombinant HW11c4, HW11c24, HW11c27 and HW11c39 were successfully expressed, further purified and functionally characterized. Both HW11c4 and HW11c27 display inhibitory activities against trypsin, chymotrypsin and kallikrein. Moreover, HW11c4 is also an inhibitor relatively specific for Kv1.1 channels. HW11c24 and HW11c39 are found to be inactive on chymotrysin, trypsin, kallikrein, thrombin and ion channels. These findings provide molecular evidence for toxin diversification of the HWTX-XI superfamily and useful molecular templates of serine protease inhibitors and ion channel blockers for the development of potentially clinical applications.     

大鼠应激耐受多系分化细胞的分离、扩增改良技术

目的 探讨体外从大鼠骨髓间充质干细胞（BMSCs）中分离应激耐受多系分化细胞（Muse cells）及其扩增培养的技术方法.方法 分离、扩增、鉴定BMSCs,应用长时间应激方法酶消化分离Muse细胞,悬浮扩增培养4d后Western blot鉴定特异性抗原.结果 第3代生长良好的BMSCs中表达CD29、CD90而不表达CD45的占细胞总数的97.89%.长时间酶消化结合悬浮培养后,以酶消化8h组细胞表达CD105显著高于其余各组（P〈0.05）,符合Muse细胞特征.结论 Muse细胞可由BMSCs经消化分离扩增获得,可望为其进一步研究应用奠定基础.     

Detection of contaminants in cell cultures,sera and trypsin

The aim of this study was standardization and application of polymerase chain reaction (PCR) for the detection of contaminants in cell cultures, sera and trypsin. Five PCR protocols were standardized to assess the presence of genetic material from mycoplasma, porcine circovirus 1 (PCV1), bovine leukemia virus (BLV) or bovine viral diarrhea virus (BVDV) in cell culture samples. PCR reactions for the genes GAPDH and beta-actin were used to evaluate the efficiency of nucleic acid extraction. The PCR protocols were applied to 88 cell culture samples from eight laboratories. The tests were also used to assess potential contamination in 10 trypsin samples and 13 fetal calf serum samples from different lots from five of the laboratories. The results showed the occurrence of the following as DNA cell culture contaminants: 34.1% for mycoplasma, 35.2% for PCV1, 23.9% for BVDV RNA and 2.3% for BLV. In fetal calf sera and trypsin samples BVDV RNA and PCV1 DNA was detected. The results demonstrated that cell culture, sera and trypsin used by different laboratories show a high rate of contaminants. The results highlight the need for monitoring cell cultures and controlling for biological contaminants in laboratories and cell banks working with these materials.     

Trypsin Activity Reduced by an Autocatalytically Produced Nonapeptide

Abstract

Trypsin, a serine protease enzyme plays a pivotal role in digestion and is autocatalytic. The crystal structure of a complex formed between porcine trypsin and an auto catalytically produced peptide is reported here. This complex shows a reduction in enzyme activity as compared to native β-trypsin. The nonapeptide has a lysine, which is recognized by Asp 189 at the specificity pocket. The auto catalytically produced native nonapeptide is bound at the active site cleft like other trypsin inhibitors but the important interactions with the oxyanion hole are absent. The peptide covers only a part of the active site cleft and hence the enzyme activity is reduced rather than being inhibited.     

血液因素对龈下菌斑BANA试验结果的影响

目的检测血液因素对慢性牙周炎龈下菌斑BANA试验敏感性、稳定性的影响。方法将不同血液成分及混有健康人血的牙龈卟咻菌菌液分别进行BANA试验。结果各检测稀释滴度的全血、冻融全血、血浆样本BANA试验反应均为阴性;当全血各稀释梯度中牙龈卟啉菌菌量大于10^5时反映结果呈现为BANA试验阳性,胰蛋白酶样酶A600nm值均大于0．20。结论BANA试验的敏感度并不受血液成分混入的影响而降低,证实试验具有较好的稳定性。     

Precision De Novo Peptide Sequencing Using Mirror Proteases of Ac-LysargiNase and Trypsin for Large-scale Proteomics

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Highlights

• •The developed Ac-LysargiNase showed higher stability and activity than before.
• •The merged spectra of the mirror peptides achieved nearly complete ion coverage.
• •pNovoM obviously increased the efficiency and accuracy of peptide sequencing.
• •The mirror enzymatic strategy achieved precision de novo sequencing on proteome scales.

     

Clinical and molecular description of the prenatal diagnosis of a fetus with a maternally inherited microduplication 22q11.2 of 2.5 Mb

Microduplications of 22q11.2 have been recently characterized as a new genomic duplication syndrome showing an extremely variable phenotype ranging from normal or mild learning disability to multiple congenital defects and sharing some overlapping features with DiGeorge/Velocardiofacial syndrome (DGS/VCFS). We report on the prenatal diagnosis of a 22q11.2 microduplication in a fetus with normal development that was referred for chromosomal analysis at 17 weeks of gestation because of advanced maternal age. Pregnancy was the result of an IVF-ICSI attempt after 4 years of infertility, mainly due to severe oligoasthenoteratospermia of the father. Amniocentesis was undertaken and cytogenetic analysis revealed an apparently normal male karyotype. Multiple Ligation-dependent Probe Amplification (MLPA) revealed a microduplication in the 22q11.2 chromosome region. Parental analysis showed that the 22q11.2 microduplication has been inherited from the otherwise healthy mother. Analysis with high resolution array-CGH showed that the size of the microduplication is 2.5 Mb and revealed the genes that are duplicated, including the TBX1 gene. The parents elected to continue with the pregnancy and the infant is now five months old and shows normal development.     

乙肝患者抗HBs独特型抗体特异性免疫复合物的检测与意义

根据用终浓度分别为35.0g/L和17.5g/L聚乙二醇沉淀循环免疫复合物,去除游离抗HBs-Ab_2,再以胰蛋白酶解离复合物的原理,建立了检测抗HBs-Ab_2-ICs的ELISA法。结果表明,38例急性乙型肝类和83例慢性活动性乙肝患者的IgG、IgM类抗HBs-Ab_2-ICs总阳性率分别为13.2％(5/38)和18.1％(15/83)。IgG、IgM类抗HBs-Ab_2-ICs检出率无显著差异(P>0.05)。实验证实乙肝患者体内存在含抗HBs-Ab_2-ICs。提示抗HBs-Ab_2尚可与抗HBs结合,抑制其中和HBV的作用而利于HBV复制。     

赤小豆抑制剂对胰蛋白酶不可逆抑制作用的动力学探讨

本文从中药赤小豆(Phaseolus Calcaratus,Roxb)中分离出一种胰蛋白酶抑制剂。通过一系列酶促反应动力学的研究表明,赤小豆抑制剂对胰蛋白酶有较强的不可逆竞争性抑制作用。其Km和ki值分别为1.43×10~(-3)mmol/L和2.4×10~(-6)mmol/L。