Measurement of plasma adenosine concentration: methodological and physiological considerations

This study tested the hypothesis that measurements of plasma adenosine concentration made on samples of blood obtained in dipyridamole and EHNA (i.e., "stopping solution") may be falsely elevated as a result of ongoing in vitro production and accumulation of adenosine during sample processing. Studies were performed with samples of anticoagulated blood obtained from anesthesized domestic swine. Adenosine concentration of ultra filtrated plasma was determined by high-pressure liquid chromatography (HPLC). The following parameters were evaluated: (i) rate of clearance of [3H]adenosine added to plasma, (ii) endogenous adenosine concentration of matched blood samples obtained in "stopping solution" alone, "stopping solution" plus EDTA, and perchloric acid (PCA), (iii) plasma and erythrocyte endogenous adenosine concentration in nonhemolyzed samples, and (iv) plasma adenosine concentration of samples hemolyzed in the presence of "stopping solution" alone or "stopping solution" plus EDTA. We observed that (i) greater than or equal to 95% of [3H]adenosine added to plasma is removed from it by formed elements of the blood in less than 20 s, (ii) plasma adenosine concentration of samples obtained in "stopping solution" alone is generally 10-fold greater than that of matched samples obtained in "stopping solution" plus EDTA, (iii) deliberate mechanical hemolysis of blood samples obtained in "stopping solution" alone resulted in substantial augmentation of plasma adenosine levels in comparison with matched nonhemolyzed specimens--addition of EDTA to "stopping solution" prevented this, and (iv) adenosine content of blood samples obtained in PCA agreed closely with the sum of plasma and erythrocyte adenosine content of samples obtained in "stopping solution" plus EDTA. The data obtained demonstrate that (i) plasma adenosine concentrations are falsely elevated in samples of blood obtained in "stopping solution" alone, and (ii) addition of EDTA to "stopping solution" blocks in vitro production and accumulation of adenosine. Finally, rapid removal of adenosine from plasma by formed elements of blood may make it difficult to employ measurements of plasma adenosine concentration to assess physiological processes even in the absence of in vitro production of the nucleoside.     

Induction of methotrexate release from rat hepatocytes in suspension by alpha-adrenergic agents: involvement of calcium and metabolic energy

Hepatocytes in suspension which have accumulated [3H]methotrexate release the antifolate compound into the medium upon exposure to alpha-adrenergic agents. In the presence of metabolic poisons, such as sodium azide, dinitrophenol, or dicumarol, the release of methotrexate is attenuated, indicating that integrity of the cellular metabolic apparatus is required for response to the hormonal stimulus. In the presence of millimolar concentrations of the organic acid, probenecid, release of cellular methotrexate may be reduced (1 mM probenecid) or eliminated (2 mM probenecid), suggesting the involvement of a "membrane carrier." Microtubule poisons such as vincristine, vinblastine, and griseofulvin do not modify epinephrine + isobutyl methyl xanthine (IBMX)-induced release of methotrexate. The involvement of calcium in release of methotrexate from the hepatocyte is substantiated by a dose-dependent response to the calcium ionophore, A23187, in the presence of calcium, with a lack of response in the absence of calcium. These effects of A23187 are not related to inhibition of methotrexate influx. Other putative "calcium antagonists" such as tetracaine, neomycin sulfate, and 3,4,5-trimethoxybenzoic acid [8-(diethylamino)octyl ester], do not interfere with epinephrine + IBMX-induced release of [3H]methotrexate, suggesting that these agents may not be effective probes of calcium flux in the liver cell.     

世居及移居高原青少年最大有氧能力的特征

本文报道海拔３４１７ｍ和４２８０ｍ地区世居藏族和移居汉族青少年运动状态下心肺功能的对比研究。结果显示：３４１７ｍ和４２８０ｍ世居藏族的最大氧耗量、无氧阈值及最大心输出量都明显大于汉族,血氧饱和度（Ｓａｏ２）随运动负荷的增加而降低。海拔３４１７ｍ藏、汉族的△Ｓａｏ２分别为７．４６％和１０．０３％,４２８０ｍ处为８．５７％和１３．７５％,最大心率随海拔升高而下降。研究提示,藏族青少年有较高的最大有氧能力,反映了他们对低氧环境的适应优势。     

Induction of the murine “W phenotype” in long-term cultures of human cord blood cells by c-kit antisense oligomers

The murine white (W) spotting locus is the site of the c-kit gene and encodes a tyrosine kinase receptor while the complementary Steel (Sl) iocus encodes its ligand. Mutations at either locus have profound effects on hematopoiesis, particularly erythroid and mast cell proliferation. We added c-kit antisense oligonucleotides to long-term suspension cultures of enriched human umbilical cord progenitor cells. This resulted in the suppression of c-kit gene expression and the preferential suppression of the generation of erythroid burst-forming cells (BFU-E) which extended over the life of the culture (3 weeks). The results provide an in vitro model of the “W phenotype” in human hematopoiesis and confirm the importance of c-kit gene function in early erythropoiesis. Because the generation of BFU-E was suppressed even after c-kit gene expression had recovered, this gene product may be critical to the erythroid commitment process. © 1993 Wiley-Liss, Inc.     

The phylogeny of the ant tribe Formicini (Hymenoptera: Formicidae) with the description of a new genus

Abstract. The holarctic ant tribe Formicini is revised, the new genus Bajcaridris described, and possible phylogenetic relationships are discussed. The subgenus Iberoformica is synonymized with Formica. A synopsis, diagnosis and keys to the genera are provided.     

Comparative Cardiac Toxicity of Anthracyclines In Vitro and In Vivo in the Mouse

Purpose

The antineoplastic efficacy of anthracyclines is limited by their cardiac toxicity. In this study, we evaluated the toxicity of doxorubicin, non-pegylated liposomal-delivered doxorubicin, and epirubicin in HL-1 adult cardiomyocytes in culture as well as in the mouse in vivo.

Methods

The cardiomyocytes were incubated with the three anthracyclines (1 µM) to assess reactive oxygen generation, DNA damage and apoptotic cell death. CF-1 mice (10/group) received doxorubicin, epirubicin or non-pegylated liposomal-doxorubicin (10 mg/kg) and cardiac function was monitored by Doppler echocardiography to measure left ventricular ejection fraction (LVEF), heart rate (HR) and cardiac output (CO) both prior to and 10 days after drug treatment.

Results

In HL-1 cells, non-pegylated liposomal-doxorubicin generated significantly less reactive oxygen species (ROS), as well as less DNA damage and apoptosis activation when compared with doxorubicin and epirubicin. Cultured breast tumor cells showed similar sensitivity to the three anthracyclines. In the healthy mouse, non-pegylated liposomal doxorubicin showed a minimal and non-significant decrease in LVEF with no change in HR or CO, compared to doxorubicin and epirubicin.

Conclusion

This study provides evidence for reduced cardiac toxicity of non-pegylated-liposomal doxorubicin characterized by attenuation of ROS generation, DNA damage and apoptosis in comparison to epirubicin and doxorubicin.     

ADAM-15/metargidin mediates homotypic aggregation of human T lymphocytes and heterotypic interactions of T lymphocytes with intestinal epithelial cells

Intestinal epithelial cells (IEC) play an immunoregulatory role in the intestine. This role involves cell-cell interactions with intraepithelial lymphocytes that may also play a role in some enteropathies. The discovery of the RGD motif-containing Protein ADAM-15 (a disintegrin and metalloprotease-15) raises the question of its involvement in these cell-cell interactions. Cell adhesion assays were performed using the Jurkat E6.1 T cell line as a model of T lymphocytes and Caco2-BBE monolayers as a model of intestinal epithelia. Our results show that an anti-ADAM-15 ectodomain antibody inhibited the attachment of Jurkat cells on Caco2-BBE monolayers. Overexpression of ADAM-15 in Caco2-BBE cells enhanced Jurkat cell binding, and overexpression of ADAM-15 in Jurkat cells enhanced their aggregation. Mutagenesis experiments showed that both the mutation of ADAM-15 RGD domain or the deletion of its cytoplasmic tail decreased these cell-cell interactions. Moreover, wound-healing experiments showed that epithelial ADAM-15-mediated Jurkat cell adhesion to Caco2-BBE cells enhances the mechanisms of wound repair. We also found that ADAM-15-mediated aggregation of Jurkat cells increases the expression of tumor necrosis factor-alpha mRNA. These results demonstrate the following: 1) ADAM-15 is involved in heterotypic adhesion of intraepithelial lymphocytes to IEC as well as in homotypic aggregation of T cells; 2) both the RGD motif and the cytoplasmic tail of ADAM-15 are involved for these cell-cell interactions; and 3) ADAM-15-mediated cell-cell interactions are involved in mechanisms of epithelial restitution and production of pro-inflammatory mediators. Altogether these findings point to ADAM-15 as a possible therapeutic target for prevention of inappropriate T cell activation involved in some pathologies.     

Autophagy as a mechanism of radiation sensitization in breast tumor cells

Current studies to define the mechanism by which vitamin D3 and analogs of vitamin D3 enhance the response to ionizing radiation in breast tumor cells suggest that these effects are mediated, in large part, through the promotion of autophagic cell death. The residual surviving cell population remains in a senescent, growth arrested state, with minimal recovery of proliferative capacity. It is becoming evident that pathways other than or in addition to apoptosis, including senescence arrest, mitotic catastrophe and autophagy, contribute to loss of self-renewal capacity in tumor cells exposed to chemotherapeutic drugs and ionizing radiation. How and why the cell chooses a particular growth arrest and/or cell death pathway remains a puzzle to be solved.