Genetic tagging of tumor cells with retrovirus vectors: clonal analysis of tumor growth and metastasis in vivo.

Retrovirus vector infection was used to introduce large numbers of unique genetic markers into tumor cell populations for the purpose of analyzing comparative changes in the clonal composition of metastatic versus that of nonmetastatic tumors during their progressive growth in vivo. The cell lines used were SP1, a nonmetastatic, aneuploid mouse mammary adenocarcinoma, and SP1HU9L, a metastatic variant of SP1. Cells were infected with delta e delta pMoTN, a replication-defective retrovirus vector which possesses the dominant selectable neo gene and crippled long terminal repeats. G418r colonies were obtained at a frequency of 4 x 10(-3). Southern blot analysis of a number of clones provided evidence of random and heritable integration of one or two copies of the proviral DNA. Clonal evolution of primary tumor growth and the nature of lineage relationships among spontaneous metastases and primary tumors were analyzed by subcutaneously injecting 10(5) cells from a pooled mixture of 3.6 x 10(2) G418r SP1HU9L or 10(4) G418r SP1 colonies into syngeneic CBA/J mice. The most striking finding was the relative clonal homogeneity of advanced primary tumors; they invariably consisted of a small number (less than 10) of distinct clones despite the fact that hundreds or thousands of uniquely marked clones had been injected. In the case of the metastatic SP1HU9L cells, the nature of these "dominant" clones varied from one tumor to another. Analysis of a number of lung metastases revealed that a proportion of them were derived from dominant primary tumor clones and were composed of one, and sometimes two, distinct progenitors. In some animals, all the lung metastases were derived from a common progenitor clone, whereas in others, each metastatic nodule had a different progenitor. The results show the following. (i) Retrovirus vector infection can be used to introduce large numbers of unique and stable clonal markers into tumor cell populations. (ii) The progeny of a very limited number of clones dominate in advanced primary tumors. (iii) Mammary carcinoma metastases are of mono- or biclonal origin. The significance of the results is discussed.     

Activation of Ca2+-dependent processes during heat shock: role in cell thermoresistance

In this brief review, it is proposed that some Ca2+-dependent processes are induced upon subjecting cells to hyperthermic temperature, and play an essential role in the final cell responses. The triggering signal does not involve external Ca2+. Instead, it is most likely to be generated by a redistribution of Ca2+ between the internal pools. A role for heat-induced Ca2+-dependent processes is supported by findings that Ca2+-active agents such as chelators, ionophores, or anticalmodulin drugs modify the cytotoxic action of hyperthermia and that some heat shock proteins are calmodulin-binding proteins. Furthermore, within minutes at hyperthermic temperature, changes are observed in the pattern of phosphoproteins suggesting that heat shock activates kinase or phosphatase activities, processes which are often mediated by Ca2+. Suggestive evidence that these phosphorylation events are determinants of cell thermoresistance is provided by the fact that one of these proteins whose phosphorylation changes rapidly upon hyperthermia is a heat shock protein (HSP28) and that the content of HSP28 is elevated not only in thermotolerant cells but also in a family of thermoresistant variants isolated after mutagenesis of Chinese hamster cells.     

蚕豆叶片发育与衰老过程中超氧物歧化酶活性与丙二醛含量变化

蚕豆植株叶片随茎节自上而下表现出明显的发育与衰老顺序,可作为衰老特征的是叶绿素和蛋白质含量明显下降。蚕豆叶中SOD活性主要定位于12 000× g离心后所得的上清液和叶绿体组分。衰老叶片的SOD总活性和叶绿体组分的相对活性都有所下降,SOD同工酶谱也发生了改变。O_2~ 产生速率随叶龄增大而稍上升;而MDA含量在叶片外观表现枯黄衰老征兆前就急剧上升。可能因为衰老叶片过氧化氢酶活性大幅度下降与SOD之间的不平衡,致使O_2~ 代谢中间产物累积而引起膜的损伤.     

GAPs for rho-related GTPases

Ras-related GTP-binding proteins (GTPases) of the rbo subfamily play important roles in regulating the organization of the actin cytoskeleton. A large number of multifunctional proteins that can stimulate their intrinsic GTPase activity have been identified. Here, we discuss the nature of such GTPase-activating proteins (GAPs) and their potential importance for cell signalling.     

Induction of vascular smooth muscle cell growth by selective activation of the thrombin receptor. Effect of heparin.

The synthetic peptide, SFLLRNPNDKYEPF, has been recently described as a peptide mimicking the new amino-terminus created by cleavage of the thrombin receptor, therefore acting as an agonist of the thrombin receptor. This peptide was a potent mitogen for rabbit arterial smooth muscle cells (SMC) and exhibited the same activity as that of native alpha-thrombin. Both compounds stimulated the proliferation of growth-arrested SMCs with half-maximum mitogenic responses at 1 nM. NAPAP, a synthetic inhibitor of the enzymatic activity of thrombin, specifically inhibited thrombin-induced SMC growth (IC50 = 0.35 +/- 0.04 microM) but was without effect on the mitogenic effect of the agonist peptide. These results therefore demonstrate that the mitogenic effect of alpha-thrombin for SMCs is intimately linked to its esterolytic activity. Heparin, which inhibited fetal calf serum-induced SMC growth, was without effect on thrombin-induced SMC growth but strongly reduced the mitogenic effect of the agonist peptide (IC50 = 32 +/- 5 micrograms/ml). This effect was not related to the anti-coagulant activity of heparin but was highly dependent on molecular mass and on the global charge of the molecule and was also observed for other sulphated polysaccharides such as pentosan polysulphate.     

Biosynthesis of lysosomal hydrolases: their synthesis in bound polysomes and the role of co- and post-translational processing in determining their subcellular distribution

By in vitro translation of mRNA’s isolated from free and membrane-bound polysomes, direct evidence was obtained for the synthesis of two lysosomal hydrolases, β-glucuronidase of the rat preputial gland and cathespin D of mouse spleen, on polysomes bound to rough endoplasmic reticulum (ER) membranes. When the mRNA’s for these two proteins were translated in the presence of microsomal membranes, the in vitro synthesized polypeptides were cotranslationally glycosylated and transferred into the microsomal lumen. Polypeptides synthesized in the absence of microsomal membranes were approximately 2,000 daltons larger than the respective unglycosylated microsomal polypeptides found after short times of labeling in cultured rat liver cells treated with tunicamycin. This strongly suggests that nascent chains of the lysosomal enzymes bear transient amino terminal signals which determine synthesis on bound polysomes and are removed during the cotranslational insertion of the polypeptides into the ER membranes. In the line of cultured rat liver cells used for this work, newly synthesized lysosomal hydrolases showed a dual destination; approximately 60 percent of the microsomal polypeptides detected after short times of labeling were subsequently processed proteolytically to lower molecular weight forms characteristic of the mature enzymes. The remainder was secreted from the cells without further proteolytic processing. As previously observed by other investigations in cultured fibroblasts (A. Gonzalez-Noriega, J.H. Grubbs, V. Talkad, and W.S. Sly, 1980, J Cell Biol. 85: 839-852; A. Hasilik and E.F. Neufeld, 1980, J. Biol. Chem., 255:4937-4945.) the lysosomotropic amine chloroquine prevented the proteolytic maturation of newly synthesized hydrolases and enhanced their section. In addition, unglycosylated hydrolases synthesized in cells treated with tunicamycin were exclusively exported from the cells without undergoing proteolytic processing. These results support the notions that modified sugar residues serve as sorting out signals which address the hydrolases to their lysosomal destination and that final proteolytic cleavage of hydrolase precursors take place within lysosome itself. Structural differences in the carbohydrate chains of intracellular and secreted precursors of cathespin D were detected from their differential sensitivity to digestion with endoglycosidases H and D. These observations suggest that the hydrolases exported into the medium follow the normal secretory route and that some of their oligosaccharides are subject to modifications known to affect many secretory glycoproteins during their passage through the Golgi apparatus.     

Experimental study of the sensitizing power of methoxyflurane]

Methyoxyflurane, a halogenated general anaesthetic, showed a net sensibilizing effect in the guinea-pig when tested experimentally by the Magnusson and Klingman technique. According to these authors classification it may be classed as of "moderate allergenicity". In addition, the sensibilisation reaction obtained is prolonged. It remains at a similar level for 2 to 7 weeks after the end of the sensibilisation phase.     

Effect of weight loss,independent of change in diet composition,on apolipoprotein AI kinetic in men with metabolic syndrome

We investigated the effect of weight loss, independent of change in diet composition, on HDL and apoAI metabolism in men with metabolic syndrome (MetS). Subjects (19 men with MetS [NCEP-ATPIII]) were fed an isoenergetic Mediterranean-style diet for 5 weeks (all foods provided). Participants then underwent a 20-week free-living period during which they were counseled to restrict energy intake, after which they were again fed an isoenergetic Mediterranean-style diet for 5 weeks. At the end of the two controlled diets, participants received a single bolus of [5,5,5-²H₃] _L-leucine, and fasting blood samples were collected over a 96 h period. ApoAI kinetic was assessed using multicompartmental modeling of the tracer enrichment data. Participants achieved a 9.1 ± 2.8% reduction in body weight (P < 0.001). Weight loss resulted in an increase in plasma HDL-cholesterol (HDL-C) concentrations of 6.0% (P = 0.059) and HDL₃-C of 7.9% (P = 0.045), attributable to a reduction in apoAI fractional catabolic rate (−7.8%; P = 0.046) with no change in apoAI production rate (2.2%; P = 0.58). These data indicate that weight loss, independent of variation in diet composition, increases plasma HDL primarily by delaying the catabolism of apoAI.     

Extent and Degree of Shoreline Oiling: Deepwater Horizon Oil Spill,Gulf of Mexico,USA

The oil from the 2010 Deepwater Horizon spill in the Gulf of Mexico was documented by shoreline assessment teams as stranding on 1,773 km of shoreline. Beaches comprised 50.8%, marshes 44.9%, and other shoreline types 4.3% of the oiled shoreline. Shoreline cleanup activities were authorized on 660 km, or 73.3% of oiled beaches and up to 71 km, or 8.9% of oiled marshes and associated habitats. One year after the spill began, oil remained on 847 km; two years later, oil remained on 687 km, though at much lesser degrees of oiling. For example, shorelines characterized as heavily oiled went from a maximum of 360 km, to 22.4 km one year later, and to 6.4 km two years later. Shoreline cleanup has been conducted to meet habitat-specific cleanup endpoints and will continue until all oiled shoreline segments meet endpoints. The entire shoreline cleanup program has been managed under the Shoreline Cleanup Assessment Technique (SCAT) Program, which is a systematic, objective, and inclusive process to collect data on shoreline oiling conditions and support decision making on appropriate cleanup methods and endpoints. It was a particularly valuable and effective process during such a complex spill.