Biosynthesis and post-translational processing of lectin-like oxidized low density lipoprotein receptor-1 (LOX-1). N-linked glycosylation affects cell-surface expression and ligand binding

LOX-1 (lectin-like oxidized low density lipoprotein receptor-1) is a type II membrane protein belonging to the C-type lectin family that can act as a cell-surface receptor for atherogenic oxidized low density lipoprotein (Ox-LDL) and may play crucial roles in atherogenesis. In this study, we show, by pulse-chase labeling and glycosidase digestion, that LOX-1 is synthesized as a 40-kDa precursor protein with N-linked high mannose carbohydrate chains (pre-LOX-1), which is subsequently further glycosylated and processed into the 48-kDa mature form within 40 min. Furthermore, when treated with an N-glycosylation inhibitor, tunicamycin, both tumor necrosis factor-alpha-activated bovine aortic endothelial cells and CHO-K1 cells stably expressing bovine LOX-1 (BLOX-1-CHO) exclusively produced a 32-kDa deglycosylated form of LOX-1. Cell enzyme-linked immunosorbent assay, flow cytometry, and immunofluorescence confocal microscopy demonstrated that the deglycosylated form of LOX-1 is not efficiently transported to the cell surface, but is retained in the endoplasmic reticulum or Golgi apparatus in tumor necrosis factor-alpha-activated bovine aortic endothelial cells, but not in BLOX-1-CHO cells. Radiolabeled Ox-LDL binding studies revealed that the deglycosylated form of LOX-1 expressed on the cell surface of BLOX-1-CHO cells has a reduced affinity for Ox-LDL binding. Taken together, N-linked glycosylation appears to play key roles in the cell-surface expression and ligand binding of LOX-1.     

Molecular cloning and expression profile of Xenopus calcineurin A subunit(1)

We have cloned a cDNA encoding a catalytic subunit of calcineurin (CnA) expressed in Xenopus oocytes. The deduced amino acid sequence indicates 96.3% and 96.8% identities with the mouse and human CnAalpha isoforms, respectively. Xenopus CnA (XCnA) RNA and protein are expressed as maternal and throughout development. Recombinant XCnA protein interacted with calmodulin in the presence of Ca(2+). Deletion of calmodulin binding domain and auto-inhibitory domain revealed calcium independent phosphatase activity, thereby showing that XCnA is likely to be modulated by both calmodulin and calcium.     

Self-amplification system for recombinant adeno-associated virus production

A recently reported system for recombinant adeno-associated virus (rAAV) production does not require infection of a helper virus and depends on the transfection with a huge amount of three plasmids: AAV-vector, AAV-helper, and adenovirus-helper plasmids. Toward simplifying rAAV production, as a first step, we tested the use of the rAAV itself instead of the AAV-vector plasmid as a source of rAAV DNA and determined the optimal timing of infection and dose of the input rAAV. When 293 cells were infected just after transfection with 100 particles/cell of rAAV, irrespective of the purity, CsCl-purified or crude, up to 2000 particles/cell of rAAV were produced (9- to 20-fold self-amplification), a yield comparable to that obtained by an adenovirus-free transfection. These results indicate that infection of rAAV can greatly reduce the amount of plasmid DNA for a large-scale transfection. This strategy will also be useful when applied to packaging cell lines inducibly expressing Rep and Cap proteins.     

中国棉铃虫核型多角体病毒DNA聚合酶基因的克隆和序列分析

应用谷实夜蛾核型多角体病毒（ＨｚＳＮＰＶ）ＤＮＡ聚合酶基因ＨｉｎｄⅢ／ＰｓｔⅠ３５９６ｂｐ片段作探针,经Ｓｏｕｒｔｈｅｒｎｂｌｏｔ杂交,克隆了中国棉铃虫核型多角体病毒（ＨａＳＮＰＶ）完整的ＤＮＡ聚合酶基因,大小约为３．４ｋｂ。限制性内切酶分析表明,ＨａＳＮＰＶＤＮＡ聚合酶基因限制性内切酶图谱与ＨｚＳＮＰＶ相似。用双脱氧链终止法测定该基因部分核苷酸序列（８０５ｂｐ）,推导出编码区２０６ａ。序列同源性比较显示,ＨａＳＮＰＶＤＮＡ聚合酶与ＨｚＳＮＰＶ之间具有高度的同源性;与ＬｄＭＮＰＶ、ＡｃＭＮＰＶ、ＢｍＳＮＰＶ、ＣｆＭＮＰＶ和ＯｐＭＮＰＶ也具有一定的同源性     

蚯蚓体内一种纤溶酶原激活剂(e-PA)的部分性质研究

从赤子爱胜蚓（Ｅｉｓｅｎｉａｆａｅｔｉｄａ）中纯化出的一种纤溶酶原激活剂（ｅ－ＰＡ）在纤维蛋白平板上可表现出三种活性,分别记为：ＣＦＰｇ,ｕＣＦＰｇ和ｕＣＦ．为更好了解各种活性与ｅ－ＰＡ的纤溶能力的关系,考察了在ＳＤＳ和不同抑制剂存在下各种活性的变化．结果表明,ＳＤＳ可以增强ＣＦＰｇ活性且使得ｅ－ＰＡ变得对一些抑制剂更敏感;ｌｅｕｐｅｐｔｉｎ,ｃｈｙｍｏｓｔａｔｉｎ,ｐｅｐｓｔａｔｉｎ,ａｐｒｏ－ｔｉｎｉｎ,ｐｈｅｎｙｌｍｅｔｈｙｌｓｕｌｆｏｎｙｌｆｌｕｏｒｉｄｅ（ＰＭＳＦ）和ｄｉｔｈｉｏｔｈｒｅｉｔｏｌ（ＤＴＴ）对ｕＣＦ没有影响;ｐｅｐ－ｓｔａｔｉｎ能增强ＣＦＰｇ和ｕＣＦＰｇ活性,Ｅ－６４（一种巯基抑制剂）能增强ｕＣＦＰｇ和ｕＣＦ活性．这些现象说明不能简单将ｅ－ＰＡ归结为丝氨酸蛋白酶或巯基蛋白酶．此外又以纤溶酶原为底物,分析了ｅ－ＰＡ在体外降解天然蛋白质的肽键特异性,结果表明：ｅ－ＰＡ可以切割碱性氨基酸,小的中性氨基酸及Ｍｅｔ的羧基端,同时ｅ－ＰＡ确能将纤溶酶原切割为纤溶酶;这一结论为ｅ－ＰＡ有可能成为新型溶栓药物提供了生化基础．     

蚯蚓体内一种纤溶酶原激活剂(e-PA)对ATEE的降解

赤子爱胜蚓（Ｅｉｓｅｎｉａｆｅｔｉｄａ）体内的一种纤溶酶原激活剂（ｅ－ＰＡ）能够降解人工合成底物Ｎ－乙酰－Ｌ－酪氨酸乙酯（ＡＴＥＥ）,该降解反应的最适ｐＨ为８．５,而且在０．２ｍｏｌ／ＬＮａ２ＨＰＯ４中的活性要强于在０．０５ｍｏｌ／ＬＴｒｉｓ－ＨＣｌ（ｐＨ８．５）中．分别测定了ｅ－ＰＡ的大小亚基及全酶在０．２ｍｏｌ／ＬＮａ２ＨＰＯ４与０．０５ｍｏｌ／ＬＴｒｉｓ－ＨＣｌ（ｐＨ８．５）两种体系中的Ｋｍ和Ｋｃａｔ．结果表明,在０．２ｍｏｌ／ＬＮａ２ＨＰＯ４中,全酶的ＡＴＥＥ活性远远高于大小亚基单独的ＡＴＥＥ活性,而在０．０５ｍｏｌ／ＬＴｒｉｓ－ＨＣｌ（ｐＨ８．５）中则没有这种现象．从蛋白质结构的角度对这一结果作了解释．用不同抑制剂和ｅ－ＰＡ作用,结果表明,ｐｅｐｓｔａｔｉｎ,Ｅ－６４和ＥＤＴＡ对ｅ－ＰＡ的ＡＴＥＥ活性都有不同程度的抑制,这一点与ｅ－ＰＡ的ＢＡＥＥ活性不同．     

Stimulation of rat hepatocyte proliferation in vitro and in vivo by factors derived from the bovine small intestinal mucosa

Summary A factor with a molecular weight of less than 1 kDa in the mucosa of the bovine small intestine (low molecular weight factor or LMW factor) stimulated DNA synthesis in rat hepatocytes in primary culture. This factor only showed its activity when it was added with a larger factor with a molecular weight of 30 kDa that was also found in the same tissue (high molecular weight factor or HMW factor). The LMW factor probably acts to enhance the action of a hepatotrophic growth factor, since EGF and HGF can substitute for the HMW factor. The action of the LMW factor was not due to the actions of low molecular weight substances such as norepinephrine, estradiol, triiodothyronine, and putrescine, which enhance the action of EGF or HGF, since substantial amounts of these substances were not found in the extract. When intraperitoneally administered into rats, after two-thirds hepatectomy, the LMW factor enhanced hepatocyte proliferation without the administration of the HMW factor. In the regenerating liver, a hepatotrophic growth factor(s), which acts synergistically with the LMW factor, might be properly provided, but the supply of the LMW factor might be below the level that maximally stimulates hepatocyte proliferation.     

Nitric oxide inhibits larval settlement in Amphibalanus amphitrite cyprids by repressing muscle locomotion and molting

Nitric oxide (NO) is a universal signaling molecule and plays a negative role in the metamorphosis of many biphasic organisms. Recently, the NO/cGMP (cyclic guanosine monophosphate) signaling pathway was reported to repress larval settlement in the barnacle Amphibalanus amphitrite. To understand the underlying molecular mechanism, we analyzed changes in the proteome of A. amphitrite cyprids in response to different concentrations of the NO donor sodium nitroprusside (SNP; 62.5, 250, and 1000 μM) using a label‐free proteomics method. Compared with the control, the expression of 106 proteins differed in all three treatments. These differentially expressed proteins were assigned to 13 pathways based on KEGG pathway enrichment analysis. SNP treatment stimulated the expression of heat shock proteins and arginine kinase, which are functionally related to NO synthases, increased the expression levels of glutathione transferases for detoxification, and activated the iron‐mediated fatty acid degradation pathway and the citrate cycle through ferritin. Moreover, NO repressed the level of myosins and cuticular proteins, which indicated that NO might inhibit larval settlement in A. amphitrite by modulating the process of muscle locomotion and molting.     

Probucol-Induced α-Tocopherol Deficiency Protects Mice against Malaria Infection

The emergence of malaria pathogens having resistance against antimalarials implies the necessity for the development of new drugs. Recently, we have demonstrated a resistance against malaria infection of α-tocopherol transfer protein knockout mice showing undetectable plasma levels of α-tocopherol, a lipid-soluble antioxidant. However, dietary restriction induced α-tocopherol deficiency is difficult to be applied as a clinical antimalarial therapy. Here, we report on a new strategy to potentially treat malaria by using probucol, a drug that can reduce the plasma α-tocopherol concentration. Probucol pre-treatment for 2 weeks and treatment throughout the infection rescued from death of mice infected with Plasmodium yoelii XL-17 or P. berghei ANKA. In addition, survival was extended when the treatment started immediately after parasite inoculation. The ratio of lipid peroxidation products to parent lipids increased in plasma after 2 weeks treatment of probucol. This indicates that the protective effect of probucol might be mediated by the oxidative stressful environment induced by α-tocopherol deficiency. Probucol in combination with dihydroartemisin suppressed the proliferation of P. yoelii XL-17. These results indicated that probucol might be a candidate for a drug against malaria infection by inducing α-tocopherol deficiency without dietary α-tocopherol restriction.