Amplification of multicistronic plasmids in the human 293 cell line and secretion of correctly processed recombinant human protein C

We have constructed multicistronic vectors containing the cDNAs for murine dihydrofolate reductase (DHFR), hygromycin phosphotransferase (HyPR), and human protein C (HPC), an antithrombotic factor. Using a sequential selection protocol with hygromycin (Hy) and methotrexate (MTX), we demonstrate the selective amplification of the murine dhfr cDNA in the adenovirus-transformed human kidney cell line 293, and the coamplification of the cDNA for HPC. Such recombinant 293 cell lines secreted HPC at levels as high as 25 micrograms/10(6) cells/day. In addition, we found that the complex vitamin K-dependent posttranslational modification of gamma-carboxylation of glutamate was not limiting at these high secretion levels, although the proteolytic processing of the protein was slightly reduced. Further, the HPC secreted from the gene-amplified cell lines had full anticoagulant activity when compared to plasma-derived HPC.     

心肌细胞间的电耦联

心脏是由无数个独立的心肌细胞通过缝隙连接形成的一个功能性合胞体。心肌细胞间的电耦联是心脏“全或无”性动作电位传导和机械收缩的先决条件。缺氧、缺血、药物、细胞内外离子浓度的改变以及激素等因素可直接或间接地影响心肌细胞间的电耦联,从而导致心脏功能的改变。     

向日葵离体孤雌生殖的超微结构研究

本文是研究未受精胚珠培养诱导的孤雌生殖过程超微结构变化的首次报道。向日葵(Heliaanthus annuus L.)的卵细胞在离体条件下被激活,发生细胞核移位、极性丧失、细胞器增多并转变成活动状态、液泡化程度增大、合点端形成细胞壁等一系列变化,预示即将启动孤雌生殖。孤雌生殖的原胚具有若干显著特征,如极性颠倒、有自体吞噬活动、壁的自由生长、游离核分裂等。对这些现象作了初步的讨论。     

青海藏族青少年骨龄与生长发育关系研究

本文报告了青海省境内,世居在海拔3000-4000米地区的728名7-18岁健康藏族青少年学生的手、腕部骨骼发育情况,对骨化中心出现和骨骺愈合求出了50%出现年龄,并对骨龄与青春期身高突增的关系及与月经初衬潮的关系进行了分析。     

Design and implementation of a particle concentration fluorescence method for the detection of HIV-1 protease inhibitors.

A critical step in the replicative cycle of the human immunodeficiency virus HIV-1 involves the proteolytic processing of the polyprotein products Prgag and Prgag-pol that are encoded by the gag and pol genes in the viral genome. Inhibitors of this processing step have the potential to be important therapeutic agents in the management of acquired immunodeficiency syndrome. Current assays for inhibitors of HIV-1 protease are slow, cumbersome, or susceptible to interference by test compounds. An approach to the generation of a rapid, sensitive assay for HIV-1 protease inhibitors that is devoid of interference problems is to use a capture system which allows for isolation of the products from the reaction mixture prior to signal quantitation. In this paper, we describe a novel method for the detection of HIV-1 protease inhibitors utilizing the concept of particle concentration fluorescence. Our approach involves the use of the HIV-1 protease peptide substrate Ser-Gln-Asn-Tyr-Pro-Ile-Val which has been modified to contain a biotin moiety on one side and a fluorescein reporter molecule on the other side of the scissile Tyr-Pro bond. This substrate is efficiently cleaved by the HIV-1 protease and the reaction can be readily quantitated. Known inhibitors of the protease were readily detected using this new assay. In addition, this approach is compatible with existing instrumentation in use for broad screening and is highly sensitive, accurate, and reproducible.     

Transfer of IncP Plasmids to Extremely Acidophilic Thiobacillus thiooxidans

The broad-host-range IncP plasmids RP4, R68.45, RP1::Tn501, and and pUB307 were transferred directly to extremely acidophilic Thiobacillus thiooxidans from Escherichia coli by conjugation at frequencies of 10^-5 to 10^-7 per recipient. The ability of T. thiooxidans to receive and express the antibiotic resistance markers was examined. The plasmid RP4 was transferred back to E. coli from T. thiooxidans at a frequency of 1.0 × 10^-3 per recipient.     

肥皂草素类核糖核蛋白体失活蛋白的纯化及其性质研究

 利用强阳离子交换柱从Saponaria officinalis L种子中分离出一种肥皂草素(Saporin)成分。它在无细胞体系中显示了较强的抑制蛋白合成的活性,与抗体连接后能特异性杀伤靶细胞。     

PHA激活的脐血T细胞条件培养液对人骨髓CFU-c的抑制

本文研究了PHA刺激18小时收获的脐血T细胞条件培养液(PHA-TCM)对正常人骨髓CFU-c的影响。结果显示PHA-TCM能够显著抑制CFU-c的生长,这种抑制与PHA-TCM浓度有关。并发现经PHA-TCM作用后M型集落比例明显降低。PHA-TCM中未检出IFN和IL-2活性。进一步研究证实,PHA-TCM中CFU-c抑制活性是一种对酸碱敏感对热相对不敏感的蛋白质,其分子量大于10,000道尔顿。     

A SPOROPHYTE CELL WALL PROTEIN OF THE RED ALGA PORPHYRA PURPUREA (RHODOPHYTA) IS A NOVEL MEMBER OF THE CHYMOTRYPSIN FAMILY OF SERINE PROTEASES1

A complementary DNA (cDNA) clone from a Porphyra purpurea (Roth) C. Agardh sporophyte-specific subtracted cDNA library was found to encode a protein similar to serine proteases of the chymotrypsin class. The encoded protein contains a typical signal peptide and is particularly similar to chymotrypsins in the regions surrounding the active site residues and the activation site where cleavage of the propeptide occurs. In addition, the six cysteine residues characteristic of chymotrypsins are conserved. However, two of the three residues of the active site His/Asp/Ser charge relay triad have been replaced, indicating that the protein is unlikely to have peptidase activity. Northern hybridization confirmed that this cDNA is derived from an abundant, sporophyte-specific messenger RNA (mRNA). The presence of signal peptide on the encoded protein and the abundance of its mRNA suggested that this protein might be localized in the cell wall. Consequently, sporophyte cell walls were isolated and a major protein having a molecular weight similar to that estimated for the encoded protein was purified. N-terminal sequence analysis indicated that this cell wall protein is identical to that encoded by the cDNA with the amino terminus of the mature protein beginning at the activation site. This cell wall structural protein appears to have evolved from a chymotrypsin-like progenitor but has been adapted to bind cell wall proteins and/or polysaccharides rather than to cleave proteins.     

Monoclonal Antibodies Against the Human Metalloprotease EC 3.4.24.15 Label Neurofibrillary Tangles in Alzheimer's Disease Brain

Abstract: Alzheimer's disease is characterized neuropathologically by the presence of neuritic and amyloid plaques, vascular amyloid, and neurofibrillary tangles in specific brain areas. The main constituent of amyloid deposits is amyloid β protein, a 40–42 amino acid proteolytic product of the amyloid β-precursor protein. In our search for proteases that can generate the N-terminus of amyloid β protein (β-secretases), we discovered a thiol-dependent metalloprotease that was identified, by peptide sequencing, as metalloendopeptidase EC 3.4.24.15. In vitro, the metalloprotease cleaves the methionine-aspartic acid bond in a 10 amino acid synthetic peptide, indicating that it could generate the N-terminus of amyloid β protein, and generates amyloidogenic fragments from full-length recombinant amyloid β-precursor protein. Mouse monoclonal antibodies produced against a unique synthetic peptide from the metalloprotease labeled various monkey tissues as detected by western blots and immunohistochemistry. Unexpectedly, two monoclonal antibodies, IVD6 and IIIF3, immunolabeled strongly intracellular neurofibrillary tangles, neurites of senile plaques, and neuropil threads, but not "ghost" tangles or amyloid in sections taken from Alzheimer's disease brain. This finding provides further evidence for the metalloprotease's relevance to Alzheimer's disease pathology, although the connection between tangle staining and the formation of amyloid β protein remains to be elucidated.