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41.
High levels of expression of full length human pro-alpha 2(V) collagen cDNA in pro-alpha 2(V)-deficient hamster cells 总被引:2,自引:0,他引:2
A full length cDNA encoding human pro-alpha 2(V) collagen was constructed. Partial sequencing of the cDNA and primer extension analysis of mRNA from fibroblasts found that pro-alpha 2(V) mRNA differs from the mRNAs of other fibrillar collagens in the increased length of its 5'-untranslated region. The pro-alpha 2(V) cDNA was placed downstream of the human cytomegalovirus immediate early promoter/regulatory sequences for expression studies in cultured Chinese hamster lung cells. These cells have been shown previously to synthesize large quantities of pro-alpha 1(V) homotrimers as their only collagenous product. Transfection resulted in a number of clonal cell lines that express human alpha 2(V) RNA at levels comparable to, and in some cases greater than, levels found in normal human skin fibroblasts. Pro-alpha 2(V) chains produced in the majority of clonal lines were of sufficient quantity to complex all available endogenous pro-alpha 1(V) chains. Chimeric heterotrimers, composed of hamster alpha 1(V) and human alpha 2(V) chains in a 2:1 ratio, were stable to pepsin digestion and were found predominantly associated with the cell layer. Surprisingly, pro-alpha 2(V) chains, in excess to pro-alpha 1(V) chains, were found in the extracellular matrix and, in much greater abundance, in media. These chains were pepsin sensitive, indicating that pro-alpha 2(V) chains can be secreted as nonstable homotrimers or as free chains. 相似文献
42.
Tyrosinase plays a pivotal role in the synthesis of melanin pigment synthesis on skin utilizing tyrosine as a substrate. Melanin is responsible for the protection against harmful ultraviolet irradiation, which can cause significant pathological conditions, such as skin cancers. However, it can also create esthetic problems when accumulated as hyperpigmented spots. Various skin-whitening ingredients which inhibit tyrosinase activity have been identified. Some of them, especially ones with natural product origins, possess phenolic moiety and have been employed in cosmetic products. Semi-synthetic and synthetic inhibitors have also been developed under inspiration of the natural inhibitors yet some of which have no phenolic groups. In this review, tyrosinase inhibitors with natural, semi-synthetic and synthetic origins are listed up with their structures, activities and characteristics. Further, a recent report on the adverse effect of a natural melanin synthesis inhibitor which was included in skin-whitening cosmetics is also briefly discussed. 相似文献
43.
SUMO化是一种重要的蛋白质翻译后修饰,对植物正常生长发育不可或缺。到目前为止已筛选到上千个可能的SUMO底物,但由于SUMO化修饰水平普遍很低,其生物学功能研究相对较少。该文详细描述了检测蛋白SUMO化修饰的常用方法,包括体外和体内SUMO化实验,以及SUMO化修饰位点的检测方法,旨在为深入研究植物蛋白SUMO化修饰提供技术支持。 相似文献
44.
45.
S C Lee J Y Shew F H Chang M J Chou C S Yang 《Proceedings of the National Science Council, Republic of China. Part B, Life sciences》1984,8(2):143-147
Protein kinase activity has been found in hepatitis B virions (Dane particles) purified from the plasma of hepatitis B surface antigen carriers [Albin, C., and Robinson, W.S. (1980) J. Virol. 34, 297-302]. Dane particles were purified from the pooled, HBeAg-positive plasma. When this preparation was incubated with [gamma 32P]ATP in the presence of 10mM MnCl2 and 0.5% NP-40 for 15 seconds at 30 degrees C, several phosphorylated polypeptides of 20,000, 42,000, 48,000, 50,000 and 56,000 daltons were detected in sodium dodecyl sulfate-polyacrylamide gels. When the Dane particles were incubated with [gamma 32P]ATP, 10 mM MnCl2, and 0.5% NP-40 in the presence of human hepatoma cell (J-5) particulate fraction at 30 degrees C, 15 seconds, the 42,000, 48,000 and 50,000 daltons phosphorylated polypeptides were not found. When human peripheral blood lymphocytes particulate fraction was incubated with Dane particles under the same conditions, no change of Dane particle phosphorylated polypeptides was detected. Previous publications [Albin, C., and Robinson, W.S. (1980) J. Virol. 34, 297-302; Gerlich, W.H. et al. (1982) J. Virol. 42, 761-766] showed that when hepatitis B core particles purified from hepatoma tissues contained protein kinase activity, only phosphorylated polypeptide was 20,000 daltons. Our data suggested that when Dane particles were put in an environment of hepatoma cells (or tissues), the protein kinase could only phosphorylate selected polypeptides in these particles. 相似文献
46.
Formation of Virus Assembly Intermediate Complexes in the Cytoplasm by Wild-Type and Assembly-Defective Mutant Human Immunodeficiency Virus Type 1 and Their Association with Membranes 总被引:4,自引:0,他引:4
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We have previously identified two distinct forms of putative viral assembly intermediate complexes, a detergent-resistant complex (DRC) and a detergent-sensitive complex (DSC), in human immunodeficiency virus type 1 (HIV-1)-infected CD4(+) T cells (Y. M. Lee and X. F. Yu, Virology 243:78-93, 1998). In the present study, the intracellular localization of these two viral assembly intermediate complexes was investigated by use of a newly developed method of subcellular fractionation. In wild-type HIV-1-infected H9 cells, the DRC fractionated with the soluble cytoplasmic fraction, whereas the DSC was associated with the membrane fraction. The DRC was also detected in the cytoplasmic fraction in H9 cells expressing HIV-1 Myr- mutant Gag. However, little of the unmyristylated Gag and Gag-Pol proteins was found in the membrane fraction. Furthermore, HIV-1 Gag proteins synthesized in vitro in a rabbit reticulocyte lysate system in the absence of exogenous lipid membrane were able to assemble into a viral Gag complex similar to that of the DRC identified in infected H9 cells. The density of the viral Gag complex was not altered by treatment with the nonionic detergent Triton X-100, suggesting a lack of association of this complex with endogenous lipid. Formation of the DRC was not significantly affected by mutations in assembly domains M and L of the Gag protein but was drastically inhibited by a mutation in the assembly I domain. Purified DRC could be disrupted by high-salt treatment, suggesting electrostatic interactions are important for stabilizing the DRC. The Gag precursor proteins in the DRC were more sensitive to trypsin digestion than those in the DSC. These findings suggest that HIV-1 Gag and Gag-Pol precursors assemble into DRC in the cytoplasm, a process which requires the protein-protein interaction domain (I) in NCp7; subsequently, the DRC is transported to the plasma membrane through a process mediated by the M domain of the matrix protein. It appears that during this process, a conformational change might occur in the DRC either before or after its association with the plasma membrane, and this change is followed by the detection of virus budding structure at the plasma membrane. 相似文献
47.
48.
Hongchi Yu Yang Shen Jingsi Jin Yingying Zhang Tang Feng 《Cell Adhesion & Migration》2018,12(1):56-68
Hepatocellular carcinoma (HCC) is a subtype of malignant liver cancer with poor prognosis and limited treatment options. It is noteworthy that mechanical forces in tumor microenvironment play a pivotal role in mediating the behaviors and functions of tumor cells. As an instrumental type of mechanical forces in vivo, fluid shear stress (FSS) has been reported having potent physiologic and pathologic effects on cancer progression. However, the time-dependent mechanochemical transduction in HCC induced by FSS remains unclear. In this study, hepatocellular carcinoma HepG2 cells were exposed to 1.4 dyn/cm2 FSS for transient duration (15s and 30s), short duration (5 min, 15 min and 30 min) and long duration (1h, 2h and 4h), respectively. The expression and translocation of Integrins induced FAK-Rho GTPases signaling events were examined. Our results showed that FSS endowed HepG2 cells with higher migration ability via reorganizing cellular F-actin and disrupting intercellular tight junctions. We further demonstrated that FSS regulated the expression and translocation of Integrins and their downstream signaling cascade in time-dependent patterns. The FSS downregulated focal adhesion components (Paxillin, Vinculin and Talin) while upregulated the expression of Rho GTPases (Cdc42, Rac1 and RhoA) in long durations. These results indicated that FSS enhanced tumor cell migration through Integrins-FAK-Rho GTPases signaling pathway in time-dependent manners. Our in vitro findings shed new light on the role of FSS acting in physiologic and pathological processes during tumor progression, which has emerged as a promising clinical strategy for liver carcinoma. 相似文献
49.
A cDNA library was prepared from poly(A) mRNA extracted from 9-day hamster-yolk-sac erythroid cells. Two clones containing inserts coding for embryonic beta-like z or y globin-chains were isolated. Their identity was confirmed by (a) translation of hybrid selected mRNAs and (b) nucleotide sequence analysis of the inserts and comparison to the embryonic beta-like globin genes of Balb/c mouse. Availability of sequences for embryonic and adult globin cDNAs will aid in investigations of the molecular mechanisms of the globin switch in hamster YSEC. 相似文献
50.
Blocking of acidosis-mediated apoptosis by a reduction of lactate dehydrogenase activity through antisense mRNA expression. 总被引:3,自引:0,他引:3
D Jeong T S Kim J W Lee K T Kim H J Kim I H Kim I Y Kim 《Biochemical and biophysical research communications》2001,289(5):1141-1149
Lactic acid produced from the cells is a potential cause of extra- and intracellular acidification. Due to scarce technical tools, lactic acid that leads to acidification could not be reduced and direct evidence of the relationship between metabolic lactate and apoptosis has not yet been elucidated. In this study, we designed a cellular pH regulation system in CHO cells by a reduction of lactate dehydrogenase (LDH) activity through LDH antisense mRNA expression. This inhibited lactate production and, therefore, acidification of the cytosol. Under HCO3(-)-buffered growth conditions, both the parent CHO cells and the engineered CHO cells maintained their extracellular pH and intracellular pH fairly well. However, upon acidification of the cytosol, only the parent CHO cells underwent apoptosis under HCO3(-)-free conditions. In fact, we observed a number of apoptosis-related events only in control cells, including mitochondrial dysfunction, cytochrome c release, and an increase in caspase-3 enzymatic activity. 相似文献