共查询到20条相似文献,搜索用时 15 毫秒
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Screening methods for chemotherapeutic agents usually rely on the cytotoxic properties of the drugs. However, agents that inhibit invasion may have more efficacy and cause fewer side effects. Various cellular invasion assays have been used to evaluate these types of compounds, including the modified Boyden chamber, monolayer wound models and Matrigel outgrowth assays. In this report, we have combined the use of the Matrigel outgrowth assay with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) visualization and cell viability dye to visualize invasive cells on Matrigel without magnification. Extraction of the dye's formazan byproduct allows cell viability to be assessed. Using several invasive and noninvasive cell lines, the utility of the method for various target cells was verified. Several established chemotherapeutic agents were also screened for their anti-invasive and/or cytotoxic effects when cultured on Matrigel. Our results suggest that this method may be an easy, inexpensive and nonradioactive alternative for both enumerating cells on Matrigel and screening various tumor cell lines treated with chemotherapeutic agent to look for compounds with noncytotoxic but anti-invasive properties. 相似文献
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JS Lewis DW McCarthy TJ McCarthy Y Fujibayashi MJ Welch 《Canadian Metallurgical Quarterly》1999,40(1):177-183
We have evaluated Cu-diacetyl-bis(N4-methylthiosemicarbazone) (Cu-ATSM), an effective marker for the delineation of hypoxic but viable tissue, in vitro in the EMT6 carcinoma cell line under varying degrees of hypoxia and compared it with the flow tracer 64Cu-pyruvaldehyde-bis(N4-methylthiosemicarbazone) (Cu-PTSM) and the hypoxic tracer 18F-fluoromisonidazole (MISO). We have also compared the uptake of Cu-ATSM and Cu-PTSM in vivo and ex vivo in a murine animal model bearing the EMT6 tumor. METHODS: Uptake of 64Cu-ATSM, 64Cu-PTSM and 18F-MISO in vitro into EMT6 cells was investigated at the dissolved oxygen concentrations of 0, 1 x 10(3), 5 x 10(3), 5 x 10(4) and 2 x 10(5) ppm. Biodistribution performed at 1, 5, 10, 20 and 40 min compared 64Cu-ATSM with 64Cu-PTSM in BALB/c mice bearing EMT6 tumors. To determine long-term retention of 64Cu-ATSM, biodistribution was also performed at 1, 2 and 4 h. Ex vivo autoradiography of tumor slices after co-injection of 60Cu-PTSM (60Cu, T1/2 = 23.7 min) and 64Cu-ATSM (64Cu, t1/2 = 12.7 h) into the same animal was performed. RESULTS: After 1 h, 64Cu-ATSM was taken up by EMT6 cells: 90% at 0 ppm, 77% at 1 x 10(3) ppm, 38% at 5 x 10(3) ppm, 35% at 5 x 10(4) ppm and 31% at 2 x 10(5) ppm. 18F-MISO also showed oxygen concentration dependent uptake, but with lower percentages than 64Cu-ATSM. 64Cu-PTSM showed 83%-85% uptake into the cells after 1 h, independent of oxygen concentration. Biodistribution data of 64Cu-ATSM and 64Cu-PTSM showed optimal tumor uptake after 5 and 10 min, respectively (0.76% injected dose (ID)/organ for 64Cu-ATSM and 1.11%ID/organ for 64Cu-PTSM). Ex vivo imaging experiments showed 60Cu-PTSM uniform throughout the EMT6 tumor, but heterogeneous uptake of 64Cu-ATSM, indicative of selective trapping of 64Cu-ATSM into the hypoxic tumor cells. CONCLUSION: Cu-ATSM exhibits selectivity for hypoxic tumor tissue both in vivo and in vitro and may provide a successful diagnostic modality for the detection of tumor ischemia. 相似文献
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H Schr?der S Warren MJ Bargetzi SV Torti FM Torti 《Canadian Metallurgical Quarterly》1993,347(6):664-666
The effect of N-acetyl-L-cysteine on the cytotoxicity of tumor necrosis factor-alpha was investigated in cultured bovine pulmonary artery endothelial cells and L929 mouse tumor cells. In endothelial cells, a 72-h incubation with tumor necrosis factor-alpha (100 ng/ml) reduced the number of viable cells to 27% of control. Simultaneous incubation with N-acetyl-L-cysteine (0.5-5 mmol/l) protected endothelial cells from tumor necrosis factor-alpha-mediated cytotoxicity and increased viability in a concentration-dependent fashion to 69% of control. Under the same conditions, a 72-h incubation with tumor necrosis factor-alpha (100 ng/ml) reduced the number of viable L929 tumor cells to 31% of control. However, this cytotoxic response remained unaltered in the presence of N-acetyl-L-cysteine (0.5-5 mmol/l). Similar results were obtained when using a lower concentration of tumor necrosis factor-alpha (50 ng/ml). These findings demonstrate protection from tumor necrosis factor-alpha-mediated toxicity by N-acetyl-L-cysteine in endothelial cells but not in a tumor cell line. It is concluded that N-acetyl-L-cysteine might serve as a therapeutic agent to limit the vascular toxicity of tumor necrosis factor-alpha without affecting its antineoplastic activity. 相似文献
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Mitomycin C and hyperthermia are both toxic to chronically hypoxic EMT6 tumor cells. Combinations of this drug and heat were tested in vitro in normally aerated and chronically hypoxic EMT6 mouse mammary tumor cells to establish whether greater than additive cytotoxicity could be achieved by combined treatment. Cell survival was measured at four concentrations of mitomycin C (0.01, 0.1, 1.0, and 10 microM) at 37 degrees or at elevated temperatures (41, 42, and 43 degrees) for durations of 1, 2, 3, and 6 hr. At 42 degrees, exposure to mitomycin C for 3 and 6 hr produced a 2- to 3-fold increase in hypoxic tumor cell kill at all drug concentrations over that expected for strict additivity. A 15-fold enhancement in the kill of hypoxic tumor cells was obtained at 1.0 and 10 microM mitomycin C at 43 degrees for 6 hr of exposure. Under most conditions, additivity was observed for the antibiotic and heat in oxygenated cells, except at 43 degrees with 0.01 and 0.1 microM mitomycin C following 3 and 6 hr of treatment, conditions under which a 5- to 10-fold potentiation of tumor cell kill was obtained. The rate of formation of reactive metabolites from mitomycin C under anaerobic conditions in EMT6 cell-free preparations was measured. A 30 to 50% increase in alkylating activity was observed at elevated temperatures, suggesting that the enhanced cytotoxicity of mitomycin C with heat toward hypoxic cells may, in part, be due to an increase in activation of the drug. 相似文献
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Nucleic acid reactive antibodies have been reported to inhibit various nucleic acid mediated functions in cell free systems. These antibodies were also shown to inhibit the growth of transformed cells in culture due to the high rate of endocytosis in transformed cells as compared to normal cells. In this report, we have tested the possibility of nucleic acid reactive antibodies inhibiting the growth of tumor cells in vivo. The life span of mice bearing Dalton's lymphoma ascites tumor cells was increased, when they were immunized with conjugates of guanosine-BSA, GMP-BSA and tRNA-MBSA complex before transplanting the tumor cells. A similar effect was also observed when mice were injected intraperitoneally with antibodies to guanosine or GMP along with the tumor cells. The specificity was ascertained, as immunization with non-specific antigens did not show any significant effect on tumor bearing mice. The results shows that nucleic acid reactive antibodies inhibit the growth of tumor cells in vivo. 相似文献
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The proteinase inhibitor set in skeletal muscle is poorly characterized at present. This study was aimed to investigate in mouse skeletal muscle 1) the tissue-associated counterpart, if any, of serum protease inhibitors (which may also play antiproteolytic functions in tissues) and 2) calpastatin, a tissue inhibitor of calcium-activated neutral proteases (calpains). Triton-extracts were prepared from muscle homogenates of mice, which had been perfused extensively with phosphate buffered saline (PBS) (under deep anesthesia) to remove blood inhibitors. Among various inhibitors tested, the following muscle-associated inhibitors were identified by western-blotting: alpha-2-macroglobulin (185, 165, 35 kDa), alpha-1-antitrypsin (52 kDa), inter-alpha-trypsin inhibitor (220, 180 kDa) and calpastatin (70 kDa). Combined light microscope and confocal immunohistochemical experiments revealed that, in all muscles examined (soleus, plantaris, extensor digitorum longus) the above specific immunoreactivities were localized outside the muscle fibers (in periendomysium, blood vessel wall) as well as within them. Inter-alpha-trypsin inhibitor, however, completely lacked the intracellular localization. This wide distribution of proteinase inhibitors suggests that numerous muscular structures may be normally protected from unwanted proteolysis, thus providing an essential background for further studies on pathological models with altered proteolysis (m. dystrophy, denervation atrophy, etc.). 相似文献
8.
Z Illés T Kondo K Yokoyama T Ohashi T Tabira T Yamamura 《Canadian Metallurgical Quarterly》1999,162(3):1811-1817
Although clonal expansion of autoimmune T cells has been reported in multiple sclerosis (MS), very limited information is available on specificities, clonal size, or activation state of the expanded clones. Here we address the issue of clonal expansion by using a novel technique demonstrating clonotypes defined by single-strand conformation polymorphism of TCR beta-chain cDNAs. Examination of activated T cells (CD3+CD25+) isolated from the peripheral blood of MS revealed limited numbers (20 approximately 82) of expanded clones defined by single-strand conformation polymorphism clonotype. To estimate the Ag specificities of dominant clonotypes in the activated T cells, these samples were examined in parallel with Th1 T cell clones specific for myelin basic protein or proteolipid protein (PLP) derived from the same patients. Analysis of two patients demonstrated that the dominant clonotypes would contain those specific for myelin basic protein or PLP. Although the majority of the clonotypes could be detected only transiently, a PLP95-116-specific clonotype was found to persist for over 1 yr. Thus, single-strand conformation polymorphism clonotype analysis allows us to monitor the kinetics of given T cell clones in vivo and could provide useful information for designing clonotype (Id)-specific manipulation of human diseases such as MS. 相似文献
9.
We describe a unique in vivo technique which addresses the multifactorial function of the heart, i.e., simultaneous measurement of myocardial ion transport (two mini-electrode systems to measure K+e and Ca2+e), energy metabolism (NADH fluorescence to measure NADH redox state), and coronary flow (laser-Doppler perfusion) using a multiprobe assembly (MPA) which contains transducers for all measurements. The MPA (which is 6 mm in diameter) was applied to the external surface of the heart in an open chest dog model. To test MPA function, myocardial ischemia was produced by application of a balloon occluder to the left anterior descending coronary (LAD) artery, and hypoxia was produced by changing the inspired O2-N2 ratio until the PaO2 was 20-30 torr. The MPA simultaneously monitored changes in ion flux, heart metabolism, and tissue perfusion during pathophysiological intervention. 相似文献
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C Ratomponirina S Gobaille Y Hodé V Kemmel M Maitre 《Canadian Metallurgical Quarterly》1998,346(2-3):331-337
Five days of gamma-hydroxybutyrate (GHB) administration (3 x 500 mg kg(-1) day(-1) i.p.) to rats resulted in a significant decrease in the density of GHB receptors measured in the whole rat brain without modification of their corresponding affinity. Similar administration of (-)-sulpiride (2 X 100 mg kg(-1) day(-1) i.p. for 5 days) induces an up-regulation of GHB receptors without change in their dissociation constants (Kd). Haloperidol (2 X 2 mg day(-1) i.p. for 5 days) showed no effect. Administered chronically via osmotic minipumps directly into the lateral ventricles, (-)-sulpiride (60 microg day(-1) for 7 days) and GHB (600 microg day(-1) for 7 days) up-regulated and down-regulated rat brain GHB receptors, respectively. Finally, in a mouse hybridoma cell line (NCB-20 cells) expressing GHB receptors, the treatment of these cells with 1 mM GHB, 100 microM (-)-sulpiride or 1 mM GABA decreases, increases and induces no change, respectively, in the density of GHB receptors after 3 days of treatments. These results indicate that chronic GHB treatment modifies the expression of its receptor and that sulpiride also induces plastic changes in GHB receptors perhaps via antagonistic properties. 相似文献
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P Janik W Roszkowski HL Ko S Szmigielski G Pulverer J Jeljaszewicz 《Canadian Metallurgical Quarterly》1980,98(1):51-58
The cytostatic action of Propionibacterium granulosum was studied in a mouse sarcoma in vivo. Kinetic analysis of tumor cells 28 days after tumor implantation and systemic immunotherapy showed that the cell cycle time was identical in both treated and untreated tumors. P. granulosum treatment resulted in a marked prolongation of the S phase and shortening of the G1 phase of the cell cycle. A pronounced drop in the number of labeled interphases and the reduction of the growth fraction were observed in tumors obtained from mice given injection of P. granulosum. Cloning efficiency of tumor cells from P. granulosum treated animals was quantitatively similar to that of control animals and only differences in the size of lung colonies were observed. 相似文献
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K Kinoshita I Ikai T Gomi M Kanai S Tsuyuki T Hirose Y Kawai A Yamauchi T Inamoto Y Inomata K Tanaka Y Yamaoka 《Canadian Metallurgical Quarterly》1998,29(2):300-305
OBJECTIVES: To determine the influence of race or ethnicity on serum prostate-specific antigen (PSA) levels and PSA density (PSAD) in a population of healthy men without clinically evident prostate cancer. METHODS: This retrospective study was conducted between January 1988 and January 1993. The serum PSA levels were measured in 859 men (586 African Americans, 142 whites, and 131 Hispanics) who were participants in a prostate cancer screening program or had urinary symptoms suggestive of prostate gland pathology. All men underwent a detailed clinical examination, including digital rectal examination, serum PSA determination, and transrectal ultrasound (TRUS). None of the subjects included had clinical or TRUS evidence of prostate cancer (furthermore, 283 men were pathologically proved to be cancer-free by prostate biopsies). Serum PSA levels and PSA densities as a function of each individual's ethnic background were determined. RESULTS: The mean serum PSA level in African Americans was 2.1 ng/mL, which was significantly higher than that of whites (mean PSA of 1.53 ng/mL) and Hispanics (mean PSA of 1.83 ng/mL) (P = 0.003). Similar differences among the three groups were observed in PSA density (the mean PSAD was 0.078, 0.057, and 0.065 for African Americans, whites, and Hispanics, respectively). A separate analysis for the biopsy-negative men was performed, and the findings were consistent with the observations for the entire study group. After adjustment for age and prostate volume, the differences remained statistically significant. CONCLUSIONS: Among men without evidence of prostate cancer, African Americans have higher serum PSA levels and PSA densities than do whites or Hispanics. Race or ethnicity was an independent factor that affected serum PSA levels even after adjustment for age and prostate volume. 相似文献
13.
Legionella pneumophila is an aquatic bacterium and is responsible for Legionnaires' disease in humans. Free-living amoebae are parasitized by legionellae and provide the intracellular environment required for the replication of this bacterium. In low-nutrient environments, however, L. pneumophila is able to enter a non-replicative viable but nonculturable (VBNC) state. In this study, L. pneumophila Philadelphia I JR 32 was suspended in sterilized tap water at 10(4) cells/ml. The decreasing number of bacteria was monitored by CFU measurements, acridine orange direct count (AODC), and hybridization with 16S rRNA-targeted oligonucleotide probes. After 125 days of incubation in water, the cells were no longer culturable on routine plating media; however, they were still detectable by AODC and by in situ hybridization. The addition of Acanthamoeba castellanii to the dormant bacteria resulted in the resuscitation of L. pneumophila JR 32 to a culturable state. A comparison of plate-grown legionellae and reactivated cells showed that the capacity for intracellular survival in human monocytes and intraperitoneally infected guinea pigs, which is considered a parameter for virulence, was not reduced in the reactivated cells. However, reactivation of dormant legionellae was not observed in the animal model. 相似文献
14.
GJ Czarnota MC Kolios H Vaziri S Benchimol FP Ottensmeyer MD Sherar JW Hunt 《Canadian Metallurgical Quarterly》1997,23(6):961-965
The densities of feline epidermal dendritic cells expressing CD18, MHC class II and CD1a antigens were determined for four anatomical locations in 19 cats of European breed in blind conditions. The densities (+/- SD) of CD1a+ Langerhans cells in the skin of the abdominal wall (269 +/- 68 cells/mm2), the back (363 +/- 19), the internal side of the ear (572 +/- 30) and the external side of the ear (502 +/- 32) were significantly different, with young and old animals displaying less stained cells than adults. No significant differences in the mean densities were found with regard to sex, colour or antibody used. 相似文献
15.
The behavior of spleen cells from tumor-bearing mice, vis-à-vis isologous tumor cells, was investigated by means of an in vivo adoptive neutralization test. C3H/eB mice were challenged with tumor cells from a chemically induced fibrosarcoma. Spleens from these animals were removed at weekly intervals following tumor inoculation, mixed with tumor cells, and tested for their influence on tumor growth in syngeneic recipient mice. Two phases in the reactivity of spleen cells from tumor-bearing mice were clearly distinguishable. In a first stage of tumor growth, these mice yielded specific tumor-inhibitory cells conferring protection. Subsequently, the protective activity declined leading to a second phase characterized by tumor enhancement. Both protective and enhancing activities were shown to be mainly dependent on the presence of T cells. 相似文献
16.
I Ohizumi S Tsunoda K Taniguchi H Saito K Esaki K Koizumi H Makimoto Y Wakai J Matsui Y Tsutsumi S Nakagawa N Utoguchi Y Ohsugi T Mayumi 《Canadian Metallurgical Quarterly》1998,77(4):561-566
An industrial erythromycin production strain of Saccharopolyspora erythraea spp. was used to demonstrate that careful genetic engineering can significantly improve productivity. The chromosomally integrated Vitreoscilla hemoglobin gene (vhb) was shown to enhance the final titer of erythromycin by some 70% compared to the original S. erythraea spp. Overall, specific erythromycin yields were about 2.5 g of erythromycin/g of total protein for S. erythraea::vhb but <1 for the S. erythraea spp. The maximum rates of biosynthesis were 57.5 mg of erythromycin/(L/h) and 24.3 mg/(L/h) for the recombinant strain S. erythraea::vhb and S. erythraea spp., respectively. Overall space-time yield was 100% higher for the S. erythraea::vhb fermentation (1.1 g of erythromycin/(L/day)) than for the S. erythraea spp. fermentation (0. 56 g of erythromycin/(L/day)). The genetic stability of the recombinant strain was high, and no selective pressure was needed throughout the cultivations. Expression of functional Vitreoscilla hemoglobin throughout the cultivations was verified by CO difference spectrum assays. 相似文献
17.
SH Devoto E Melan?on JS Eisen M Westerfield 《Canadian Metallurgical Quarterly》1996,122(11):3371-3380
We have examined the development of specific muscle fiber types in zebrafish axial muscle by labeling myogenic precursor cells with vital fluorescent dyes and following their subsequent differentiation and fate. Two populations of muscle precursors, medial and lateral, can be distinguished in the segmental plate by position, morphology and gene expression. The medial cells, known as adaxial cells, are large, cuboidal cells adjacent to the notochord that express myoD. Surprisingly, after somite formation, they migrate radially away from the notochord, becoming a superficial layer of muscle cells. A subset of adaxial cells develop into engrailed-expressing muscle pioneers. Adaxial cells differentiate into slow muscle fibers of the adult fish. We have named the lateral population of cells in the segmental plate, lateral presomitic cells. They are smaller, more irregularly shaped and separated from the notochord by adaxial cells; they do not express myoD until after somite formation. Lateral presomitic cells remain deep in the myotome and they differentiate into fast muscle fibers. Thus, slow and fast muscle fiber types in zebrafish axial muscle arise from distinct populations of cells in the segmental plate that develop in different cellular environments and display distinct behaviors. 相似文献
18.
A noncytotoxic neutralizing monoclonal antibody (mAb), 26-2F, to human angiogenin (Ang), a potent inducer of neovascularization, has been reported to prevent or delay the establishment of HT-29 human tumor xenografts in athymic mice. In the present study the tumor model was modified to increase sensitivity to Ang antagonists to facilitate further investigations and comparisons of their capacity to inhibit tumor growth. An increase in the percentage of tumor-free mice from 10-25% to 65% is observed in this modified model after treatment with mAb 26-2F. An additional neutralizing mAb, 36u, that interacts with a different epitope on Ang similarly prevents the appearance of tumors, both alone and in combination with mAb 26-2F. In those tumors that develop in mice treated with these agents, the number of vascular elements is reduced. Actin, an Ang antagonist that unlike the mAbs binds both human and mouse Ang, also prevents the establishment of tumors while exhibiting no toxic effects at daily doses > 50 times the molar amount of circulating mouse Ang. Ang antagonists also inhibit the appearance of tumors derived from two other Ang-secreting human tumor cell lines--i.e., A549 lung adenocarcinoma and HT-1080 fibrosarcoma. These results demonstrate that inhibition of the action of Ang is an effective therapeutic approach for the treatment of malignant disease. 相似文献
19.
Y Dubaquié R Looser U Fünfschilling P Jen? S Rospert 《Canadian Metallurgical Quarterly》1998,17(20):5868-5876
The mechanism of chaperonin-assisted protein folding has been mostly analyzed in vitro using non-homologous substrate proteins. In order to understand the relative importance of hsp60 and hsp10 in the living cell, homologous substrate proteins need to be identified and analyzed. We have devised a novel screen to test the folding of a large variety of homologous substrates in the mitochondrial matrix in the absence or presence of functional hsp60 or hsp10. The identified substrates have an Mr of 15-90 kDa and fall into three groups: (i) proteins that require both hsp60 and hsp10 for correct folding; (ii) proteins that completely fail to fold after inactivation of hsp60 but are unaffected by the inactivation of hsp10; and (iii) newly imported hsp60 itself, which is more severely affected by inactivation of hsp10 than by inactivation of pre-existing hsp60. The majority of the identified substrates are group I proteins. For these, the lack of hsp60 function has a more pronounced effect than inactivation of hsp10. We suggest that homologous substrate proteins have differential chaperonin requirements, indicating that hsp60 and hsp10 do not always act as a single functional unit in vivo. 相似文献
20.
The displacement of small amounts of tritiumlabbelled antagonists or agonists by increasing amounts of unlabelled antagonists in mouse brain in vivo offered the possibility of characterizing properties of opiate receptors in the intact animal. The displacement effect was stereospecific, saturable and dependent upon the affinity of the substance investigated. At brain concentrations of 0.3 nM, 75% of 3H-diprenorphine, 60% of 3H-naltrexone and 50% of 3H-naloxone were displaced by high amounts of the respective unlabelled drug. Comparison of the in vivo data with receptor binding in vitro revealed similar results in respect to binding sites and receptor affinity. The displacement was different in various brain areas. The time course of the displacement was also different for the various substances used and seems to reflect differences in the speed of association and dissociation to and from the receptors. The displacement of 3H-etorphine by naltrexone could be correlated with the reversal of analgesia. 相似文献