Trypan Blue Dye Uptake Research Articles

Commiphora myrrha stimulates insulin secretion from mouse and human islets of Langerhans

Ethnopharmacological relevanceTraditionally plant-based remedies such as Commiphora myrrha (CM) have been used as an ayurvedic medicine to treat diabetes mellitus in some region of Arabia and Africa. Previous reports have shown that CM reduced blood glucose levels and increased insulin concentrations in animal models of diabetes in vivo. However, the exact mechanisms by which CM improved glycemic control in these animals are not fully understood. We hypothesized that CM may have a direct insulinotropic activity on β-cells to increase insulin secretion. Aim of the studyThe direct effects of CM were investigated using MIN6 β-cells and isolated mouse and human islets in static and perifusion insulin secretion experiments. Isolated mouse and human islets were used to investigate the rate and pattern of CM-induced insulin secretion. Materials and methodsThe effect of CM on insulin secretion was assessed by static and perifusion experiments using MIN6 cells, a mouse-derived β-cell line, and primary mouse and human islets. The effects of CM on cell viability and membrane integrity of MIN6 cells and mouse islets were assessed using an ATP viability assay and a trypan blue exclusion test. The mRNA expression profiles of preproinsulin and Pdx1, a major β-cell transcription factor, were determined by quantitative RT-PCR following chronic exposure to CM. ResultsExposing MIN6 cells to a CM resin solution (0.5–10 mg/ml) caused a concentration-dependent increase in insulin secretion in a static setting. Similarly, incubating mouse islets to CM (0.1–10 mg/ml) resulted in stimulation of insulin secretion in a concentration-dependent manner. CM concentrations at ≤ 2 mg/ml were not associated with reduction in cell viability nor with reduction in cell membrane integrity. However, higher concentrations of CM were accompanied with marked uptake of trypan blue dye and cell death. In a perifusion setting, CM (2 mg/ml) caused rapid and reversible increases in insulin secretion from both mouse and human islets at both sub-stimulatory and stimulatory glucose levels. The stimulatory effect of CM on insulin secretion did not change the total insulin content of β-cells nor the mRNA expression of preproinsulin and Pdx1. ConclusionsThese data indicate that aqueous CM resin solution has a direct stimulatory effect on β-cells without compromising plasma membrane integrity. CM stimulates insulin secretion from MIN6 cells, a mouse-derived β-cell line, and isolated primary mouse and human islets in vitro at both sub-stimulatory and stimulatory glucose concentrations. The mechanism by which CM may induce insulin secretion is most likely due to a stimulation of insulin granules release rather than insulin synthesis.

Iodine-131 induces cell death by downregulation of antiapoptotic genes in MCF-7 human adenocarcinoma cells

Aims: Radioiodine ( 131 I) is the most common radionuclide which possesses favorable nuclear characteristics for targeted therapy in cancer management. There are several therapeutic radiopharmaceuticals labeled with 131 I used in clinics, but the basic mechanism describing the cause of induced cell death is limited in literature. Hence, the aim of the present study is to find a plausible mechanism of cellular toxicity and involvement of antiapoptotic gene in induction of cell death due to 131 I. Materials and Methods: The effect of 131 I on cell death was studied by incubating MCF-7 cell line with different amount of 131 I radioactivity for 6 h followed by washing and extended the incubation for 48 h. Cells were harvested and cell viability was assessed by lactate dehydrogenase (LDH) release and trypan blue dye uptake. Apoptosis study was carried out with enzyme-linked immunosorbent assay method. Reverse transcriptase polymerase chain reaction was carried out to find expression of antiapoptotic genes, viz., BCL-2 and BCL XL . Results: It was found that release of LDH was Dose and time dependent, and 35% cell death was estimated by trypan blue with 37 MBq of 131 I radioactivity at 48 h of incubation. Enrichment factor of apoptotic DNA was 3.2 with 37 MBq of 131 I at 48 h. Densitometric analysis of BCL-2 and BCL XL showed that there is downregulation of genes expression, which confirmed apoptotic cell death. Conclusions: 131 I induces apoptosis in MCF-7 cell line by the downregulation of antiapoptotic gene BCL-2 and BCL XL when exposed for longer time periods.

Antibacterial and Anti-Acanthamoebic Properties of Catha Edulis (Khat)

Infectious diseases remain a significant threat to human health, contributing to more than 17 million annual deaths thus indicating an urgent to identify novel molecules for antimicrobial chemotherapy. Here, the antimicrobial activities of aqueous crude extracts of Catha edulis (khat widely used in Africa and southern parts of Arabia) were tested against a panel of microorganisms including Gram-positive bacteria (Bacillus magaterium, Micrococcus luteus), Gram-negative bacteria (Escherichia coli, Brevundimonas diminuta), yeast (Aspergillus varicoluor, Penicillum solitum, Penicillum brevicompactum) and the protist (Acanthamoeba castellanii) in vitro. At 100 μg, C. edulis extracts exhibited potent antibacterial activity against B. diminuta (19 mm ± 2.3), B. magaterium (16 mm ± 0.7) and M. luteus (22 mm ± 3.1) but not against E. coli and yeast (A. varicoluor, P. solitum, P. brevicompactum). Notably, C. edulis extracts showed amoebicidal effects (>50 % reduction in amoeba numbers of the original inoculum) as evidenced by the uptake of Trypan blue dye. The remaining sub-population of A. castellanii remained viable but cultures remained static over longer incubations. These findings show that C. edulis extract possess selective antibacterial properties. For the first time, it is shown that C. edulis extract exhibit anti-Acanthamoebic properties. Further studies are needed to identify active components and assess their clinical relevance.

Modulation of cellular radiation responses by 2-deoxy-D-glucose and other glycolytic inhibitors: Implications for cancer therapy

2-Deoxy-D-glucose (2-DG), a glycolytic inhibitor, was observed earlier to increase DNA, chromosomal, and cellular damage in tumor cells, by inhibiting energy-dependent repair processes. Lonidamine (LND) selectively inhibits glycolysis in cancer cells. It damages the condensed mitochondria in these cells, impairing thereby the activity of hexokinase (predominantly attached to the outer mitochondrial membranes). It inhibits repair of radiation-induced potentially lethal cellular damage in HeLa and Chinese hamster (HA-1) cells. However, other than a preliminary study on human glioma (BMG-1) cells in this laboratory, the effects of LND on radiation-induced cytogenetic damage have not been reported earlier. These studies were carried out to investigate the effects of LND and 2-DG on cell proliferation, viability, and radiation response in the same human glioma cell line, under identical conditions. The respective drug concentrations were selected on the basis of earlier studies. Human glioma (U373MG) cells were grown in the presence of LND or 2-DG for 2 days. Proliferation response and viability of U373MG human glioma cells were studied by cell counts and uptake of trypan blue dye. Radiosensitization (increase in micronuclei formation) was studied after short-term (4 h postirradiation) drug treatments. Both the drugs (1) inhibited proliferation response in a concentration-dependent manner; (2) did not induce micronuclei formation in the unirradiated cells; and (3) significantly increased radiation-induced micronuclei formation at nontoxic concentrations. These data suggest that the short-term presence of either lonidamine or 2-DG-at clinically relevant and nontoxic concentrations-could increase the treatment response of malignant gliomas at optimum radiation doses, reducing thereby the side effects of radiotherapy.

Reversible Oxidation of ERK-directed Protein Phosphatases Drives Oxidative Toxicity in Neurons

Oxidative stress links diverse neuropathological conditions that include stroke, Parkinson's disease, and Alzheimer's disease and has been modeled in vitro with various paradigms that lead to neuronal cell death following the increased accumulation of reactive oxygen species. For example, immortalized neurons and immature primary cortical neurons undergo cell death in response to depletion of the antioxidant glutathione, which can be elicited by administration of glutamate at high concentrations. We have demonstrated previously that this glutamate-induced oxidative toxicity requires activation of the mitogen-activated protein kinase member ERK1/2, but the mechanisms by which this activation takes place in oxidatively stressed neurons are still not fully known. In this study, we demonstrate that during oxidative stress, ERK-directed phosphatases of both the serine/threonine- and tyrosine-directed classes are selectively and reversibly inhibited via a mechanism that is dependent upon the oxidation of cysteine thiols. Furthermore, the impact of ERK-directed phosphatases on ERK1/2 activation and oxidative toxicity in neurons was tested in a neuronal cell line and in primary cortical cultures. Overexpression of the highly ERK-specific phosphatase MKP3 and its catalytic mutant, MKP3 C293S, were neuroprotective in transiently transfected HT22 cells and primary neurons. The neuroprotective effect of the MKP3 C293S mutant, which enhances ERK1/2 phosphorylation but blocks its nuclear translocation, demonstrates the necessity for active ERK1/2 nuclear localization for oxidative toxicity in neurons. Together, these data implicate the inhibition of endogenous ERK-directed phosphatases as a mechanism that leads to aberrant ERK1/2 activation and nuclear accumulation during oxidative toxicity in neurons.

Adenosine attenuates oxidant injury in human proximal tubular cells via A(1) and A(2a) adenosine receptors.

We have recently demonstrated protection against renal ischemic-reperfusion injury in vivo by A(1)- and A(2a)-adenosine receptor (AR) modulations. To further elucidate the signaling cascades of AR-induced cytoprotection against reperfusion/oxidant-mediated injury, immortalized human proximal tubule (HK-2) cells were treated with H(2)O(2). H(2)O(2) caused dose- and time-dependent HK-2 cell death that was measured by lactate dehydrogenase release and trypan blue dye uptake. Adenosine protected against H(2)O(2)-induced HK-2 cell death by means of A(1)- and A(2a)-AR activation. A(1)-AR-mediated protection involves pertussis toxin-sensitive G proteins and protein kinase C, whereas A(2a)-AR-mediated protection involves protein kinase A activation by means of cAMP and activation of the cAMP response element binding protein. Moreover, protein kinase A activators (forskolin and Sp-isomer cAMP) also protected HK-2 cells against H(2)O(2) injury. De novo gene transcription and protein synthesis are required for both A(1)- and A(2a)-AR-mediated cytoprotection as actinomycin D and cycloheximide, respectively, blocked cytoprotection. Chronic treatments with a nonselective AR agonist abolished the protection by adenosine. Moreover, chronic treatments with a nonselective AR antagonist increased the endogenous tolerance of HK-2 cells against H(2)O(2). We concluded that A(1)- and A(2a)-AR activation protects HK-2 cells against H(2)O(2)-induced injury by means of distinct signaling pathways that require new gene transcription and new protein synthesis.

In vivo/ex vivo cellular interactions with titanium and copper.

Machined, commercially pure titanium (Ti) disks were coated with approximately 400 nm copper (Cu) by physical vapor deposition or left uncoated. The kinetics of inflammatory cell recruitment, distribution and viability was evaluated around Ti, Cu, and in sham sites after 1, 3, 12, 18, 24, and 48 h in a rat subcutaneous (s.c.) model. Further analysis of the cells on implant surfaces was performed by ex vivo incubation of the disks. Ti and Cu stimulated an increased recruitment of inflammatory cells in comparison with sham sites. A markedly higher amount of cells, predominantly polymorpho-nuclear granulocytes (PMN), was detected around Cu after 18 h and onwards. More cells were found at the implant surfaces than in the surrounding exudates after 18 h. The total amount of lactate dehydrogenase (LDH), an indicator of plasma membrane injury, was higher in Cu exudates after 18 h in comparison with Ti and sham. In contrast, no differences in the proportion of dead cells (trypan blue dye uptake) were detected in the exudates. Further, LDH levels were higher around Ti than Cu during the initial 18 h of ex vivo incubation. The results of this study indicate that the early inflammatory process associated with a cytotoxic material in soft tissues is largely attributed to the induction of a markedly strong and prolonged chemotactic response. In contrast, this process is characterized by a higher amount of inflammatory cells around a biocompatible material than in sham sites, but with a transient course and total LDH similar to sham sites.

The involvement of protein kinase C in nitric oxide-induced damage to rat isolated colonic mucosal cells.

1 The role of protein kinase C (PKC) in colonic cellular injury in response to high concentrations of nitric oxide (NO) released from the donor, S-nitroso-N-acetyl-DL-penicillamine (SNAP) was investigated. 2 Addition of SNAP (0.1-1000 microM) to the cellular suspension resulted in a dose-dependent increase in the extent of damage to isolated colonic mucosal cells as assessed by Trypan blue dye uptake and release of the lysosmal enzyme, N-acetyl-beta-glucosaminidase. SNAP treatment also resulted in an increase in cellular total PKC activity. These increases were reduced or eliminated by pretreatment of the cells with the PKC antagonists staurosporine or GF 109203X or the NO scavenger, phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl 3-oxide (PTIO). 3 PKC-alpha, PKC-delta, PKC-epsilon and PKC-zeta were detected in colonic cellular lysates by immunoblotting. However, only PKC-epsilon protein was increased in response to SNAP treatment. Furthermore, SNAP treatment resulted in activation of PKC-epsilon by causing translocation of the enzyme from the cytosolic to membrane fraction of the cell. This effect was eliminated if cells were preincubated with the NO scavenger, PTIO. 4 The extent of cellular damage in response to addition of SNAP to the incubation medium was enhanced by coincubation with the PKC activator, phorbol 12-myristate 13-acetate (PMA; 1 and 10 microM). 5 PKC activity and the extent of cell damage in response to SNAP were reduced by preincubation of the cells with the peroxyl scavenger, ebselen (0.01-10 microM). 6 These data suggest that the PKC-epsilon isoform of the enzyme mediates NO-induced damage to colonic mucosal cells. This response may occur, at least in part, due to peroxynitrite formation.

The effect of a high partial pressure of carbon dioxide environment on metabolism and immune functions of human peritoneal cells—Relevance to carbon dioxide pneumoperitoneum

Objective: Our purpose was to evaluate in vitro the effect of a high partial pressure of carbon dioxide environment used in laparoscopy on metabolic and immune response of various human peritoneal cells. Study Design: Polymorphonuclear leukocytes were obtained from 5 healthy volunteers, peritoneal macrophages were obtained from the effluent of 8 patients undergoing continuous ambulatory peritoneal dialysis, and human peritoneal mesothelial cell cultures were prepared from omentum derived from 5 patients undergoing elective surgery. The cells were exposed to a laparoscopy-like environment (1 atmosphere carbon dioxide and 0.2 atmosphere oxygen), to a control gas mixture (1 atmosphere helium and 0.2 atmosphere oxygen), or air for 3 hours. After exposure to gas mixtures, cell functions were tested at various recovery periods. Results: Three hours of exposure to a high partial pressure of carbon dioxide had no effect on viability of peritoneal macrophages and human peritoneal mesothelial cells, tested by trypan blue dye uptake and lactate dehydrogenase release. A high partial pressure of carbon dioxide decreased the mitochondrial dehydrogenases activity of peritoneal macrophages and human peritoneal macrophage cells by 60%, assayed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction. High partial pressure of carbon dioxide blocked the superoxide release from activated polymorphonuclear leukocytes and the secretion of interleukin 1β from stimulated peritoneal macrophages, and human peritoneal macrophage cells were decreased by 15% and 30% and the secretion of tumor necrosis factor-α from peritoneal macrophages was suppressed by 85%. Mitochondrial activity, polymorphonuclear leukocyte function, and interleukin 1β and tumor necrosis factor-α secretion returned to normal after a recovery period of 12 to 24 hours, 4.5 hours, and 24 hours, respectively. In the control experiments exposure of cells to helium had no suppressive effect. Conclusions: Exposure of cells to a high partial pressure of carbon dioxide environment suppresses the inflammatory and metabolic responses of peritoneal cells. We suggest that this suppressive effect may contribute to the low postsurgery adhesion formation and the reduction in postoperative pain observed in laparoscopy. Nevertheless, the suppression of the immune response should also be taken into account for operations involving a high risk of bacterial dissemination. (Am J Obstet Gynecol 1998;179:1503-10.)

Ca-mediated damage to rabbit gastric mucosal cells: modulation by nitric oxide

Perturbations in cellular Ca2+ homeostasis can lead to oxidative stress whereas nitric oxide has been shown to inactive oxygen radicals. Therefore the effects of inhibition of nitric oxide (NO) synthase activity on Ca2+-mediated disruption to rabbit dispersed gastric mucosal cells have been examined. Addition of the Ca2+ ionophore A23187 (3–25 μM) to the incubation medium induced a concentration-dependent increase in cell damage is assessed by trypan blue dye uptake and decreased cellular metabolic activity as estimated by alamar blue absorbance. These responses were exacerbated by inhibition of NO synthase activity with NG-monomethyl-L-arginine (300 μM). The deleterious effects of ionophore A23187 and NG-monomethyl-L-arginine were ameliorated by addition of the NO donor S-nitroso-acetyl-penicillamine to the cell suspension. An increase in cellular Ca2+ in response to ionophore A23187 (12.5 μM) resulted in enhaced 2′7′-dichlorofluorescein fluorescence suggesting an elevation in oxidative stress. Ca2+-mediated cell injury was abolished by the oxygen radical scavengers, catalase and 2′,2′-dipyridyl. However, the cytotoxic effect of combined treatment with A23187 and NG-monomethyl-L-arginine was not reduced by administration of oxygen radical scavengers. NG-monomethyl-L-arginine treatment exacerbated the increase in cytosolic Ca2+ in response to ionophore A23187 as assessed by indo-1 fluorescence. Furthermore this increase in cytosolic Ca2+ was reduced by addition of S-nitroso-acetyl-penicillamine to the incubation medium. These data suggest that NO synthase inhibition in gastric mucosal cells exacerbates the damaging actions of the Ca2+ ionophore A23187. The increase in cell damage in response to the NO synthase inhibitor NG-monomethyl-L-arginine does not appear to be mediated by an increase in oxidative stress and may be associated in part with changes in cellular Ca2+ flux.

Ca 2+-mediated damage to rabbit gastric mucosal cells: modulation by nitric oxide

Perturbations in cellular Ca 2+ homeostasis can lead to oxidative stress whereas nitric oxide has been shown to inactive oxygen radicals. Therefore the effects of inhibition of nitric oxide (NO) synthase activity on Ca 2+-mediated disruption to rabbit dispersed gastric mucosal cells have been examined. Addition of the Ca 2+ ionophore A23187 (3–25 μM) to the incubation medium induced a concentration-dependent increase in cell damage is assessed by trypan blue dye uptake and decreased cellular metabolic activity as estimated by alamar blue absorbance. These responses were exacerbated by inhibition of NO synthase activity with N G - monomethyl- L-arginine (300 μM). The deleterious effects of ionophore A23187 and N G - monomethyl- L-arginine were ameliorated by addition of the NO donor S-nitroso-acetyl-penicillamine to the cell suspension. An increase in cellular Ca 2+ in response to ionophore A23187 (12.5 μM) resulted in enhaced 2′7′-dichlorofluorescein fluorescence suggesting an elevation in oxidative stress. Ca 2+-mediated cell injury was abolished by the oxygen radical scavengers, catalase and 2′,2′-dipyridyl. However, the cytotoxic effect of combined treatment with A23187 and N G - monomethyl- L-arginine was not reduced by administration of oxygen radical scavengers. N G - monomethyl- L-arginine treatment exacerbated the increase in cytosolic Ca 2+ in response to ionophore A23187 as assessed by indo-1 fluorescence. Furthermore this increase in cytosolic Ca 2+ was reduced by addition of S-nitroso-acetyl-penicillamine to the incubation medium. These data suggest that NO synthase inhibition in gastric mucosal cells exacerbates the damaging actions of the Ca 2+ ionophore A23187. The increase in cell damage in response to the NO synthase inhibitor N G - monomethyl- L-arginine does not appear to be mediated by an increase in oxidative stress and may be associated in part with changes in cellular Ca 2+ flux.

Spermatocyte toxicity of 2-methoxyethanol in vivo and in vitro: Requirement for an intact seminiferous tubule structure for germ cell degeneration

Cultures of isolated testicular cells are widely used for evaluating mechanisms of action of direct-acting testicular toxicants. However, for testicular cells, the expression of toxicity in vitro is frequently different from that found in vivo. 2-Methoxyethanol (ME) produces testicular lesions in rats which are characterized by pachytene spermatocyte degeneration 24 hr after dosing. As part of a study to evaluate mechanisms of male germ cell toxicants, we compared the morphological aspects of spermatocyte toxicity after in vivo exposure to ME with those found in various testicular cell culture systems after in vitro exposure to the active ME metabolite, 2-methoxyacetic acid (MAA). Immature rats were used because they respond to in vivo ME treatment in the same way as adults, but their testes are relatively enriched in pachytene spermatocytes. 24-day-old rats were given a single dose of 250 mg ME/kg body weight by gavage and the testes were evaluated 24 hr after dosing. In vitro, cultured seminiferous tubules, Sertoli-germ cell co-cultures, and enriched mixed germ cells, all prepared from 24-day-old rats, were evaluated after 24-hr in vitro exposure to 5 m m MAA (a level of MAA found after ME exposure in vivo). Testes from ME-exposed rats showed the expected pachytene spermatocyte degeneration 24 hr after dosing. Similar changes were observed in cultured seminiferous tubules after 24 hr of exposure to MAA in vitro. However, without the intact seminiferous tubule structure in vitro, the expression of MAA spermatocyte toxicity was different. In conventional Sertoli-germ cell co-cultures, spermatocyte detachment from the Sertoli cell monolayer occurred as expected, although no significant morphological degeneration was observed in these detached germ cells. Similarly, no increase in degenerating spermatocytes was noted in isolated enriched mixed germ cells after in vitro MAA exposure. Instead, toxicity in these germ cell fractions was expressed only by increased uptake of trypan blue dye, revealing an increase in plasma membrane permeability. In summary, this in vivo/in vitro comparison of the spermatocyte toxicity of ME/MAA showed that the expression of toxicity is different in the different culture architectures and that an intact seminiferous tubule structure is required for full expression of the morphological degeneration produced by ME/MAA, and suggests the usefulness of culture seminiferous tubules in future mechanistic studies.

Reduced glutathione modulates Ca(2+)-mediated damage to rabbit isolated gastric mucosal cells

In the present study, we have examined the role of reduced glutathione (GSH) in the modulation of Ca2+ ionophore A-23187-induced gastric mucosal cellular disruption. Experiments were conducted using cells isolated from rabbit oxyntic mucosa. Cell injury was assessed by release of the lysosomal enzyme acid phosphatase, by trypan blue dye uptake, and by examination of lipid peroxidation. Cellular GSH was assessed spectrophotometrically by measuring total soluble reduced thiol content. Ionophore A-23187 (3-25 microM) induced a concentration-dependent injury to mucosal cells and a concentration-dependent reduction in cellular GSH content. Removal of Ca2+ from the incubation medium abolished both the disruptive action and the GSH-lowering effect of A-23187. Gastric cellular GSH content was reduced significantly by preincubation of cells with an agent that depletes reduced sulfhydryls, diethyl maleate (DEM; 1 mM). Furthermore, DEM significantly augmented cellular disruption in response to A-23187. Similarly, incubation of cells with L-cysteine (1 mM), a stimulant of glutathione synthesis, increased mucosal cellular GSH content and decreased the cellular disruptive effect of A-23187. Exogenous addition of GSH to the cell suspension significantly reduced Ca2+ ionophore-induced cellular disruption and hastened the recovery of cellular free Ca2+ concentration ([Ca2+]) to baseline values. Similarly, exogenous GSH reduced cellular disruption produced by cyclopiazonic acid and thapsigargin, agents that increase intracellular [Ca2+] ([Ca2+]i) by inhibiting Ca2+ sequestration. These data suggest that a sustained increase in [Ca2+]i contributes to the pathogenesis of gastric mucosal cellular injury. This effect appears to be mediated by a reduction in the cellular content of GSH.

Effect of extracellular Ca2+ on indomethacin-induced injury to rabbit dispersed gastric mucosal cells

The effects of extracellular Ca2+ on indomethacin-induced disruption to rabbit dispersed gastric mucosal cells have been examined. Fundic mucosal cells were isolated, and cellular viability and disruption were assessed by the release of the lysosomal enzyme acid phosphatase and by trypan blue dye exclusion. Addition of the Ca2+ ionophore A23187 (12.5 and 25 microM) and indomethacin (0.1-10 microM) to the incubation medium induced concentration-dependent increases in enzyme marker release and trypan blue dye uptake in the cells. The resultant cellular disruption in response to incubation of cells with combinations of indomethacin and A23187 was not significantly different from that observed when the agents were administered separately. The degree of cell injury was reduced by removal of Ca2+ from the incubation medium. Similarly preincubation with the Ca2+ channel antagonist verapamil (1 microM) reduced A23187- and indomethacin-induced cell injury. Both A23187 (25 microM) and indomethacin (10 microM) treatments increased cytosolic Ca2+ concentration ([Ca2+]i). Pretreatment of cells with 16,16-dimethylprostaglandin E2 (0.1-10 microM) reduced both indomethacin (10 microM) induced cellular damage and the elevation in [Ca2+]i. These data suggest that indomethacin-induced disruption of gastric mucosal cells is dependent to some extent upon extracellular Ca2+. An inappropriate Ca2+ flux may contribute to indomethacin-induced damage to gastric mucosal cells.

Gene transfer investigations of p56-LCK function in IL-2-dependent T-cell lines: Implications for mechanisms of IL-2-signal transduction

Recently, the SRC-like non-receptor protein tyrosine kinase p56-LCK has been shown to physically associate with the interleukin-2 receptor (IL-2-R) complex and to undergo rapid elevations in its tyrosine kinase activity upon stimulation of T lymphocytes with IL-2. The functional significance of p56-LCK kinase activation for IL-2-mediated lymphocyte responses, however, has never been directly assessed. Using gene transfer approaches, we have achieved markedly elevated levels of p56-LCK kinase activity in the IL-2-dependent cytolytic T-cell line CTLL-2 and the helper line HT-2. CTLL-2 and HT-2 cells that were stably transfected with expression plasmids encoding either the normal human p56-LCK or a constitutively active version of the mouse p56-LCK kinase (LCK[Y505]) contained striking elevations in the levels of tyrosine phosphorylation on several proteins (34–36, 50–60, 62–68, 77–78, 104–110 kDa), as determined by immunoblot analysis using anti-phosphotyrosine antibodies. CTLL-2 and HT-2 LCK- and LCK(Y505F)-transfected cells remained dependent on IL-2 for their growth and survival in culture despite the findings that (i) IL-2 specifically stimulated elevations in the activity of the endogenous p56-LCK in untransfected CTLL-2 cells without affecting the activities of the other SRC-like kinases in these cells (p59-FYN, p62-YES) and that (ii) IL-2-mediated regulation of p56-LCK correlated with IL-2-driven proliferation of these T cells. Specifically, no elevation in the proliferation (DNA synthesis) or growth of these T cells was found at any of the concentrations of IL-2 examined (0.01–25 U/ml), relative to untransfected and control transfected cells. Furthermore, when cultured in the absence of IL-2, transfected T cells whose relative levels of p56-LCK activity were elevated by ≈ 20–50-fold died with the same kinetics as control cells and underwent apoptosis, as defined by uptake of trypan blue dye and DNA fragmentation assays, respectively. Taken together, these data indicate that while IL-2 can up-regulate the enzymatic activity of p56-LCK, elevated levels of p56-LCK tyrosine kinase activity are insufficient to stimulate IL-2-mediated pathways required for T-cell growth and survival. These findings thus imply the existence of other signal-transducing molecules, besides p56-LCK, that physically participate in IL-2R complexes and that are necessary for initiation of the biochemical events ultimately responsible for IL-2's pleiotropic actions on lymphocytes.

Interactions of 1,10-phenanthroline and its copper complex with Ehrlich cells

Mechanistic details of the interaction of 1,10-phenanthroline and its copper complex with Ehrlich ascites tumor cells were examined, using inhibition of cell proliferation, DNA breakage, and increased membrane permeability as indices of cellular damage. The metal chelating agent 1,10-phenanthroline (OP), the 1:05 complex of 1,10-phenanthroline and CuCl 2 [(OP) 2Cu], and CuCl 2 inhibited growth of Ehrlich ascites tumor cell monolayers during 48-h treatments by 50% at about 3.5, 2, and 70 nmol/10 5 cells/mL, respectively. (OP) 2Cu at 10 nmol/10 5 cells also enhanced uptake of trypan blue dye during 6 h of treatment, while dye uptake in OP- and CuCl 2-treated cells remained similar to controls. DNA breakage, measured by DNA alkaline elution, was produced during 1-h treatments with (OP) 2Cu at drug/cell ratios similar to those producing growth inhibition. Copper uptake was similar for both (OP) 2Cu and CuCl 2. Electron spin resonance (ESR) spectroscopy suggested that cellular ligands blind copper added as (OP) 2Cu or CuCl 2 and then undergo time-dependent reductions of Cu(II) to Cu(I) for both forms. Inhibition of (OP) 2Cu-induced single-strand scission and trypan blue uptake by scavengers of activated oxygen is consistent with participation of superoxide and H 2O 2 in both processes. In contrast, superoxide dimutase (SOD) did not reduce the magnitude of the fraction of cellular DNA appearing in lysis fractions prior to alkaline elution of (OP) 2Cu-treated cells. Dimethyl sulfoxide (DMSO) inhibited uptake of trypan blue dye did not inhibit DNA strand scission produced by (OP) 2Cu. Thus, multiple mechanisms for generation of oxidative damage occur in (OP) 2Cu-treated cells. Growth inhibition produced by OP or (OP) 2Cu, as well as the low levels of strand scission produced by OP, was not reversed by scavengers.

Ultraviolet-treated lipoproteins as a model system for the study of the biological effects of lipid peroxides on cultured cells. II. Uptake and cytotoxicity of ultraviolet-treated LDL on lymphoid cell lines

The ‘cytotoxicity’ of ultraviolet-treated low-density lipoproteins (LDL) has been investigated using cultured lymphoid cell lines from normal subjects and from a patient with receptor-negative familial hypercholesterolemia. The ultraviolettreated LDL were taken up by control lymphoblasts through the classical apo B/E-receptor pathway, while they were slowly taken up by receptor-negative lymphoblasts by non-specific endocytosis. These LDL were found highly ‘cytotoxic’ on normal lymphoblasts as demonstrated by Trypan blue dye uptake, [ 3H]thymidine incorporation, lactate dehydrogenase release and by electron microscopy. The ‘cytotoxicity’ increased progressively with the concentration of ultraviolet-treated LDL in the culture medium and with the incubation time. In contrast, lymphoblasts from familial hypercholesterolemia were not sensitive to low doses of ultraviolet-treated LDL (up to 150 μg apo-B/ml). The comparison of cells from normals and familial hypercholesterolemia showed that the ‘cytotoxic’ effect occurred subsequently to the LDL uptake, either receptor-mediated or receptor-independent. Experiments combining short-time (5 h) pulse with ultraviolet-treated LDL (labelled with [ 3H]cholesteryl oleyl ether) and a relatively long-chase period (72 h) showed: (1) a relationship between the delay for the appearance of the ‘cytotoxicity’ and the amount of ultraviolet-treated LDL taken up by the cells; and (2) the existence of a minimal dose (threshold dose) for triggering the ‘cytotoxic’ effect. The use of ‘hybrid’ LDL, prepared by partial delipidation of non-treated LDL and reconstitution by re-incorporating the neutral lipid fractions isolated from ultraviolet-treated LDL, demonstrated that the ‘cytotoxic’ effect is mainly mediated by triacylglycerols and cholesterol esters. Scanning electron microscopy showed that the most prominent morphological change resulting from the uptake of ultraviolet-treated LDL was the early blebbing of plasma membranes.

Electron paramagnetic resonance evidence that cellular oxygen toxicity is caused by the generation of superoxide and hydroxyl free radicals

Cells require molecular oxygen for the generation of energy through mitochondrial oxidative phosphorylation; however, high concentrations of oxygen are toxic and can cause cell death. A number of different mechanisms have been proposed to cause cellular oxygen toxicity. One hypothesis is that reactive oxygen free radicals may be generated; however free radical generation in hyperoxic cells has never been directly measured and the mechanism of this radical generation is unknown. In order to determine if cellular oxygen toxicity is free radical mediated, we applied electron paramagnetic resonance, EPR, spectroscopy using the spin trap 5,5′-dimethyl-1-pyrroline- N-oxide, DMPO, to measure free radical generation in hyperoxic pulmonary endothelial cells. Cells in air did not give rise to any detectable signal. However, cells exposed to 100% O 2 for 30 min exhibited a prominent signal of trapped hydroxyl radical, DMPO-OH, while cell free buffer did not give rise to any detectable radical generation. This cellular radical generation was demonstrated to be derived from the superoxide radical since the observed signal was totally quenched by superoxide dismutase, but not by equal concentrations of the denatured enzyme. It was confirmed that the hydroxyl radical was generated since in the presence of ethanol the CH 3·CH(OH) radical was formed. Loss of cell viability as measured by uptake of trypan blue dye was observed paralleling the measured free radical generation. Thus, superoxide and hydroxyl radicals are generated in hyperoxic pulmonary endothelial cells and this appears to be an important mechanism of cellular oxygen toxicity.

Schistosoma mansoni: Analysis of cercarial transformation methods

Four methods of transforming cercariae to schistosomulae in vitro in ELAC buffer (pH 7.2, 37 C, 0–6 hr incubation) were compared in relation to biochemical and ultrastructural characteristics. The transformation methods used were chemical (3 m M linoleate), mechanical (centrifuge/vortex), mechanical/chemical, and heat (incubation at 37 C). Ultrastructural characteristics examined were based on the presence or absence of glycocalyx, heptalaminate membrane, cyton granules, and nuclear condition. Two EM fixation methods were used. Biochemical parameters assayed were loss of water tolerance (uptake of trypan blue dye), eicosanoid biosynthesis (PGE, LTB 4, and 5-HETE), protein synthesis (leucine uptake), RNA synthesis (uracil and orotic acid uptake), and DNA synthesis (thymidine uptake). EM characteristics were remarkably similar for all transformation methods except heat incubation, with transformed cercariae evidencing the characteristics of schistosomulae (cyton granule migration, absence of glycocalyx and heptalaminate membrane); however, euchromatic nuclei could not be demonstrated using in vivo or in vitro transformation methods. Despite the ultrastructural similarities between transformation methods, biochemical data demonstrated that the resultant organisms were quite different. The chemical transformation method gave the highest rate of loss of water tolerance and eicosanoid production. RNA and protein synthesis were not correlated to ultrastructural changes and were highest in those organisms undergoing mechanical transformation methods, significantly higher than in those cercariae transformed by the chemical method. DNA synthesis was not demonstrated using any transformation method, although thymidine uptake did occur. Our data indicate substantial biochemical differences exist between morphologically similar organisms. Thus, experiments using any type of artificially transformed schistosomule must be interpreted with caution until additional biochemical and physiological studies on cercarial transformation are undertaken.

In vitro photosensitization of human head and neck squamous cancer cells by dihematoporphyrin-ether.

In vitro experiments were performed to evaluate the direct cytotoxic and photosensitizing effects of dihematoporphyrin-ether (DHE) on the head and neck squamous cancer cell line, UM-SCC-38. Normal fibroblasts, normal cultured keratinocytes, and the UM-MEL-1 pigmented malignant melanoma cell line were used as controls. The parameters of duration of light exposure, drug concentration, and incubation periods were studied. Uptake of DHE by squamous cancer cells, as assessed microscopically by intensity of fluorescence, was rapid and reached a dose-dependent maximum intensity within 10 minutes. Cells were irradiated with polychromatic light with an energy of one milliwatt/cm2 at a distance of 1.5 ft. Cells growing in plastic dishes were incubated in the dark for 1 to 4 hours with DHE in concentrations that ranged from 1.25 to 50 micrograms/ml. The cell monolayers were washed and irradiated for periods of time ranging from 15 to 120 min. Dose-dependent loss of cell viability could be detected by trypan blue dye uptake as early as 1 hour after radiation and continued to increase until 4 hours after light exposure. No further loss of cell viability was observed over the next 10 hours. There was no phototoxicity in the absence of DHE. UM-SCC-38 cells were more sensitive to the photosensitizing effects of DHE than were either normal fibroblasts or malignant melanoma cells. Recovery from the photosensitizing effects of DHE was observed if the DHE-containing medium was removed and the cells were incubated in the dark for periods that ranged from 1 to 14 hours before light exposure. UM-SCC-38 cells recovered more rapidly than normal fibroblasts, normal keratinocytes, or UM-MEL-1 cells.