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PubMed1998

NMR spectroscopy and MRI investigation of a potential bioartificial liver.

NMR feasibility was established for a coaxial hydrophobic-membrane bioreactor containing isolated rat hepatocytes with features designed to mimic the human liver. A novel triple-tuned NMR probe and a perfusion system controlling temperature, gas concentrations, flow-rate, and pH were used. We determined the optimum coaxial interfiber distance (i.e. diffusion distance) for maintaining hepatocyte viability in two bioreactor prototypes. Prototype no. 1 and no. 2 had diffusion distances of 500 microns and 200 microns, respectively. Cell viability was established by 31P NMR and trypan blue exclusion. Only prototype no. 2 maintained cell viability for more than 6 h, indicating the importance of diffusion distance. 31P spectra obtained over this 6 h time period were similar to in vivo spectra of rat liver. The 31P spectra were found to be more sensitive to subacute cell viability than trypan blue exclusion. In the 1H and 31P spectra, 1H2O and inorganic phosphate signals were split in two at all flow-rates, probably due to bulk magnetic susceptibility effects originating from the three bioreactor compartments. MRI was useful for quality control and determining flow dynamics, fiber integrity, and cell inoculate distribution. MRI revealed that the inner fibers were not centered in either prototype. Although an increased flow-rate did not influence spectral resolution or chemical shifts, significant degradation of MRI quality occurred above 50 mL/min. NMR spectroscopy and imaging provide valuable, real-time information on cell biochemistry and flow dynamics which can be used in development and monitoring of bioreactors designed as artificial livers.

Animals↗
PubMed1986

Triorganotin-induced cytotoxicity to rat thymus, bone marrow and red blood cells as determined by several in vitro assays.

To further investigate the immunotoxic effects of tri-n-propyltin chloride (TPTC), tri-n-butyltin chloride (TBTC) and triphenyltin chloride (TPhTC) several cytotoxicity tests with a series of trialkyltin chlorides and TPhTC were carried out, using isolated rat thymocytes as target cells. Thymocytes, cultured in a serum-supplemented medium, were exposed to organotin concentrations ranging from 0.01 to 10 microM for periods up to 30 h. Parameters such as cell count, trypan blue exclusion, chromium release, thymidine incorporation and cyclic AMP production were used to evaluate the cytotoxicity of these compounds. The more lipophilic compounds TPTC, TBTC, tri-n-hexyltin chloride (THTC) and TPhTC appeared most cytotoxic, reducing thymidine incorporation at concentrations as low as 0.05-1 microM. Membrane damage as determined by trypan blue exclusion and chromium release occurred at higher levels (1-10 microM). The water soluble homologue trimethyltin chloride (TMTC) was least effective in all test models. When phosphate-buffered saline supplemented with glucose was used as incubation medium, TBTC appeared more cytotoxic to thymocytes. Using this medium in 5-h incubations the cytotoxicity of TBTC to thymus, bone marrow and red blood cells was compared. Bone marrow cells were slightly less sensitive than thymocytes, while red cells were relatively resistant. In conclusion, of the triorganotin compounds especially the lipophilic homologues are cytotoxic in vitro.

Animals↗
PubMed2005

Transscleral-RPE permeability of PEDF and ovalbumin proteins: implications for subconjunctival protein delivery.

PURPOSE: To investigate the in vitro, ex vivo, and in vivo transscleral-retinal pigment epithelium (RPE) permeability of PEDF and structurally related proteins for the exploration of novel routes of protein delivery to the retina. METHODS: Monkey RPE cells were cultured on permeable supports to separate apical and basal compartments. Porcine scleral tissue was placed in Ussing chambers to separate uveal and orbital compartments. Transepithelial resistance and voltage were measured by an electrical resistance system, and paracellular tracer flux was evaluated with trypan blue. Subconjunctival administration in rat eyes was by injections of soluble protein or by implantation of polyvinyl alcohol devices containing protein. Fluorescein-conjugated (Fl-) PEDF and ovalbumin were determined by spectrofluorometry, laser scanning, immunoblotting, epifluorescence, and confocal microscopy. Permeability was assessed by fluoresceinated-protein flux. RESULTS: Transepithelial resistance, impermeability to trypan blue, and confocal microscopy confirmed functional and structural tight junction formation of RPE cells cultured on permeable supports. Full-length Fl-PEDF and Fl-ovalbumin proteins diffused through RPE cell monolayers from either the apical or basal side. Fl-ovalbumin diffused through scleral tissues at constant rates. Subconjunctival Fl-PEDF or Fl-ovalbumin administration in vivo revealed movement of full-length protein into the choroid and retina as early as 1 hour. CONCLUSIONS: The sclera and RPE were permeable in vitro, ex vivo, and in vivo to PEDF and ovalbumin proteins. These large proteins can traverse through the sclera-RPE to reach the retina. In addition, these data prompt the proposal that subconjunctival protein delivery may represent a feasible and minimally invasive route for PEDF administration in the clinic.

Animals↗
PubMed1989

[Protective effects of nicorandil against catecholamine induced myocardial damage under hypoxia].

Direct myocardial protective effects of Nicorandil (NR) against hypoxic injury were evaluated using an in vitro model of isolated, adult rat myocytes. Ca++-tolerant, trypan blue negative, viable cells were suspended for 1 hour in a hypoxic (Po2 20 mmHg) Eagle's MEM culture medium. Myocytes were then divided into 4 groups and incubated for 3 hours either without (control) or with norepinephrine (NE) (10(-6)M), NR (10(-4)M) or NE+NR. Cell viability ratio (trypan blue staining method) and intracellular ATP contents were measured as indexes of cell damage. NE significantly decreased the cell viability ratio compared to that in control after 1 to 3 hours. Addition of NR prevented the reduction in viability ratio produced by NE. NR markedly attenuated intracellular ATP reduction induced by NE after 1 hour incubation. These results suggest that NR has potent direct effects against hypoxia-induced cell injury in the presence of catecholamines.

Adenosine Triphosphate↗
PubMed2005

Hepatotoxicity of alcohol-induced polar retinol metabolites involves apoptosis via loss of mitochondrial membrane potential.

Chronic alcohol consumption depletes hepatic vitamin A stores. However, vitamin A supplementation is hepatotoxic, which is further potentiated by concomitant alcohol consumption. It was suggested that polar retinol metabolites generated by alcohol-inducible cytochrome P4502E1 aggravate liver damage. However, experimental evidence supporting this hypothesis is lacking. To elucidate the effects of polar retinol metabolites on cultured HepG2 cells and primary rat hepatocytes, polar retinol metabolites were extracted from liver tissues of rats fed either an alcoholic or isocaloric control Lieber-DeCarli diet. Cell toxicity assays included morphology assessment, trypan blue exclusion test, and LDH/AST leakage. Staining for DAPI and acridine orange, FACS analysis, and Western blot for cleaved caspase-9 and -3 were used to detect apoptosis. Polar retinol metabolites caused marked cytotoxicity in a concentration- and time-dependent manner in both cell types reflected by morphological changes, a dramatic increase in trypan blue positive cells, and LDH/AST leakage. Toxicity was due to apoptosis, as demonstrated by a time-dependent increase of sub-G1 cellular events, a rapid loss of mitochondrial membrane potential, and a time-dependent activation of caspase-9 and -3. No toxicity was found with equivalent doses of the control extract from nonalcoholic rats. We demonstrate that polar retinol metabolites cause marked hepatocyte death through the induction of apoptosis.

Animals↗
PubMed1989

Immunological and nonimmunological control of severity of Trypanosoma musculi infections in C3H and C57BL/6 inbred mice.

Studies concerned with the mechanisms responsible for relative resistance or susceptibility of strains of inbred mice to Trypanosoma musculi infections are presented. Treatment with 400 rads of ionizing radiation, silica dust, or trypan blue (reticuloendothelial blocking agents) rendered C3H mice unable to control the initial maximum level of parasite growth, and the mice died of overwhelming infections. In contrast, similarly treated C57BL/6 (relatively resistant) mice controlled initial trypanosome growth as well as controls; however, the duration of infection, preceding eventual cure, was approximately doubled. Combined treatment with trypan blue and 400 rads of radiation resulted in much higher initial levels of infection in C57BL/6 mice, and about half of the mice died; the remaining mice eventually recovered after a prolonged course of infection. These results indicate that a nonimmunological mechanism, which controls initial infection, and an immunological mechanism cooperate to limit T. musculi infections in normal mice. We present results that suggest that both mechanisms are less effective in C3H than in C57BL/6 mice. The initial control of infection presumably reflects the activity of some type(s) of phagocytic effector cell; we show, however, that the initial control of infection is not an attribute of the liver Kupffer cells. Identification and characterization of the cells capable of controlling initial infection could lead to procedures for enhancing their function and, thus, to enhanced resistance to, and elimination of, trypanosome infections.

Animals↗
PubMed1987

Effects of the peptide toxin from Microcystis aeruginosa on intracellular calcium, pH and membrane integrity in mammalian cells.

Extracts of water blooms of the toxic cyanobacterium Microcystis aeruginosa showed a range of toxicities not related to their ability to lyse mammalian red cells. The HPLC-purified heptapeptide toxin (mol. wt. 1035) from Microcystis did not lyse red cells at up to 500-fold higher concentrations than that required to kill mice. This toxin (LD50 110 micrograms/kg for male mice) was used to investigate in vitro effects on isolated thymocytes, hepatocytes, mammary alveolar cells, and cultured Swiss 3T3 fibroblasts. Thymocytes were stimulated to progressive Ca2+ entry by toxin (0.1-10 micrograms/ml), reaching a peak after approx. 5 min. No deformation, intracellular pH change, Trypan Blue entry or cell lysis was seen within 60 min at 37 degrees C. Hepatocytes were grossly deformed by the toxin, with a dose/response relationship between 0.1 and 1.0 microgram/ml. No progressive Ca2+ entry was observed on toxin addition, instead a rapid rise in intracellular Ca2+, presumably from intracellular sources. No change in intracellular pH, Trypan Blue exclusion or cell lysis was observed over 60 min. Mammary alveolar cells and 3T3 fibroblasts were unresponsive to toxin at the concentrations tested. No change in protein synthesis or nucleic acid synthesis in thymocytes was observed after culture with 0.5 or 5.0 micrograms/ml toxin. It was concluded that cytoskeletal changes in deformed hepatocytes (the target cells in vivo) demonstrated the most probable cellular basis for toxicity, rather than changes in membrane permeability or cell metabolism.

Animals↗
PubMed1970

The release of radioactive nucleic acids and mucoproteins by trypsin and ethylenediaminetetra-acetate treatment of baby-hamster cells in tissue culture.

Monolayers of baby-hamster kidney cells were grown on glass in tissue culture and harvested with trypsin or EDTA in order to investigate the cell surface macromolecules removed by these cell-disaggregating agents. The release of nucleic acids from the cells during the harvesting procedure was monitored by labelling the cellular RNA with [5-(3)H]uridine and the cellular DNA with [2-(14)C]thymidine. Treatment of the cells with EDTA was found to cause an increase in the permeability of the plasma membrane with 7.6% of the cellular RNA, but less than 1% of the cellular DNA, being released. Moreover, 61% of the cells harvested with EDTA were permeable to Trypan Blue. With crude trypsin, lysis of the cell occurred with the release of similar amounts of RNA and DNA amounting to about 11% of the total cellular nucleic acid. In contrast, crystalline trypsin released only 1% of the cellular nucleic acids. Since virtually all the cells (99%) after harvesting in crystalline trypsin were impermeable to Trypan Blue, this method was suitable for obtaining cell surface macromolecules without contamination by intracellular damage. [1-(14)C]Glucosamine was incorporated by the cells only into bound hexosamines and sialic acids. [By monitoring the release of radioactivity in high-molecular-weight material in such experiments a measure of the release of macromolecules containing amino sugars was obtained.] Of the total macromolecules containing amino sugars in the cells 33%, 24% and 13% were released when the cells were harvested with crude trypsin, crystalline trypsin or EDTA respectively. Crystalline trypsin also released 39% of the total sialic acid of the cell, whereas less than 1% of the cellular sialic acid was present in the EDTA-treated fraction. It is concluded that the macromolecules containing amino sugars released with crude trypsin and EDTA are likely to be heavily contaminated with intracellular material. However, the macromolecules released by crystalline trypsin appear to come from the cell surface.

Animals↗
PubMed1998

Comparison of seven quantitative assays to assess lymphocyte cell death during HIV infection: measurement of induced apoptosis in anti-Fas-treated Jurkat cells and spontaneous apoptosis in peripheral blood mononuclear cells from children infected with HIV.

The study of apoptosis in relation to various human disease states, particularly HIV infection, has seen a tremendous increase in activity. In this article, values obtained by seven different assays, designed to quantify apoptosis and applicable to the study of HIV infection, are compared in two cell systems: (1) stimulus-induced apoptosis in Jurkat cells treated with anti-Fas antibody and (2) spontaneous apoptosis in PBMCs isolated from HIV-infected children. The methods used included measurement of cells with subdiploid DNA content, labeling of DNA strand breaks by the TUNEL reaction, annexin V surface labeling for the detection of exposed phosphatidylserine, cytoplasmic antigen labeling with the apoptosis-specific antibody Apo 2.7, detection of changes in flow cytometric light-scattering properties, trypan blue dye exclusion by light microscopy, and detection of changes in cellular chromatin by fluorescence microscopy. These methods produced well-correlated values in the Jurkat system, whereas the same set of methods produced more discrepant values in the PBMC analyses, especially in those patients with low CD4 counts. Specifically, our results showed that the trypan blue test was unacceptable for quantification of apoptosis during HIV infection, whereas TUNEL, of all the methods tested, showed excellent overall correlation in both cell systems, was highly specific, and matched microscopic observation of the cells. Although many of the methods were suited to the study of a homogeneous cell line, caution must be exercised when examining cell death in a heterogeneous cell mixture from an HIV-infected individual.

Adolescent↗
PubMed2004

Photodynamic therapy with indocyanine green complements and enhances low-dose cisplatin cytotoxicity in MCF-7 breast cancer cells.

OBJECTIVE: We investigated the effects of photodynamic therapy (PDT) combined with low-dose chemotherapy on breast cancer cells. Photodynamic treatment was administered by irradiating indocyanine green-preloaded MCF-7 cells with an IR diode laser source at 805 nm; cisplatin was used for chemotherapy. METHODS: The dose-response phenomena associated with the two treatments administered individually and together were evaluated with the following tests: trypan blue dye exclusion, 3-(4,5-dimethylthiazol)-2,5-diphenyltetrazolium bromide (MTT) assay, clonogenic survival, thymidine and methionine incorporation, and insulin-dependent and insulin-independent glucose transport. RESULTS: Viability and metabolic data demonstrated mutual reinforcement of therapeutic efficacy. However, isobolographic analysis of quantal and variable data indicated that reinforcement was additive according to trypan blue data and synergistic according to MTT data. To investigate the molecular mechanisms underlying alterations in cell proliferation and apoptosis, we evaluated (by Western blotting) the expression of proteins Bcl-2, Bax, Bcl-X(L), p21, p53, and poly(ADP-ribose) polymerase. Photodynamic treatment caused transient selective destruction of Bcl-2 and up-regulation of Bax. It also induced apoptosis in a limited fraction of cells (10-12%). Flow cytometry data showed that PDT killed mostly G(1)-phase cells, whereas cisplatin killed mostly S-phase cells. This disjointed phase-related effect may account for the favorable effects exerted by combined treatment. CONCLUSIONS: Our findings imply that low doses of cytostatic drugs may be as effective or even more effective than currently used doses if appropriately combined with PDT.

Antineoplastic Agents↗
PubMed1996

Preconditioning of primary rat neuronal cultures against ischemic injury: characterization of the "time window of protection'.

Primary rat neuronal cultures can be preconditioned against ischemic damage by several mechanisms. In the present study we established a new model system in order to characterize the "time window of protection' obtained by preconditioning of neurons with adenosine. Ischemia was simulated by exposure of the cultures to iodoacetate (100 microM) for 150 min, with a post-ischemic reperfusion period of 60 min. Ischemic injury was assessed by the release of lactic dehydrogenase (LDH) to the medium during the ischemic period and ischemia-reperfusion damage by the Trypan blue exclusion test. Exposure of the neuronal cultures to the ischemic or ischemia-reperfusion insult resulted in severe damage to the neurons, manifested for the former insult in a 5.4-fold increase in the release of LDH and for the latter insult in an 8.5-fold increase in the proportion of stained cells by the Trypan blue exclusion test. Preconditioning by short exposure (5 min) of the cultures to iodoacetic acid (simulating sublethal ischemia), or to adenosine (1 mM) and the A1 adenosine receptor agonist N6-(R)-phenylisopropyladenosine (R-PIA; 1 and 100 microM), prior to the insult, partially protected the neurons against the damage. The time-course of the development and waning of the resistance against the two insults following preconditioning exhibited different patterns. The resistance obtained against the ischemic insult developed rapidly, being maximal for all substances at 10 min (the shortest time window studied), and lasted up to 1 h for iodoacetate, 3 h for R-PIA and 24 h for adenosine. In contrast, the protection induced by adenosine and R-PIA against ischemia-reperfusion injury developed relatively slowly, being maximal at 3 h, but lasted longer, up to 48 h. At this time the time-response curve exhibited a second peak of protection. The waning of protection against the two insults was found to continue into a period of increased sensitivity to the insults. This phenomenon was more intense for preconditioning with iodoacetate, and especially against the ischemic injury. The results suggest that in the neurons, different mechanisms may mediate the adenosine-induced preconditioning against the ischemic or ischemia-reperfusion injury. In addition, the results support the possibility that the relatively long "time window of protection', induced by adenosine and R-PIA against ischemia-reperfusion insult, reflects a combination of two different preconditioning mechanisms.

Animals↗
PubMed1992

Reperfusion rather than storage injury predominates following long-term (48 h) cold storage of grafts in UW solution: studies with Carolina Rinse in transplanted rat liver.

Both storage injury and reperfusion injury have been reported in association with liver transplantation; however, which predominates is not clear. Therefore, these studies were designed to evaluate whether Carolina Rinse, which minimizes reperfusion injury following orthotopic liver transplantation in the rat, would be effective after long-term (48 h) storage of grafts in University of Wisconsin (UW) cold storage solution where sufficient time for development of storage injury exists. Livers were rinsed with either Ringer's solution or Carolina Rinse solution immediately prior to completion of implantation surgery. In the Ringer's group, 30-day survival was high following 24 h of cold storage (4/5) but was very low after 48 h (1/16). Importantly, survival was increased significantly (5/14) when grafts were rinsed with Carolina Rinse following 48 h of cold storage. In both groups, parenchymal cells appeared normal by scanning electron microscopy, excluded trypan blue, and released SGOT at values only slightly above the normal range immediately (i.e., less than 5 min) after 48 h of cold storage. However, SGOT values rose steadily during the 1st hour postoperatively following reperfusion in the Ringer's rinse group and reached levels around 1,000 U/l. In addition, nonparenchymal cells were not labelled with trypan blue following storage, but significant labelling occurred within 1 h. Both SGOT release and nonparenchymal cell injury were reduced significantly when grafts were rinsed with Carolina Rinse prior to completion of surgery. Liver injury assessed histologically 24 h postoperatively was also reduced about 50% by Carolina Rinse. Oxidative stress appeared to be involved, since radical adducts, most likely of lipid origin, were trapped during the first 5 min after reperfusion with the spin trapping technique and detected by electron paramagnetic resonance spectroscopy. Lipid radical formation was reduced nearly completely on reperfusion by Carolina Rinse. Since Carolina Rinse improved survival of liver grafts following long periods of cold storage and reduced lipid radical formation and hepatocellular injury, we concluded that a reperfusion injury rather than a storage injury predominates following orthotopic transplantation of livers stored for long periods of time in cold UW solution.

Adenosine↗
PubMed2003

Comparison of the efficacy of biodegradable and non-biodegradable scintillation liquids on the counting of tritium- and [14C]-labeled compounds.

The widespread use of H and 14C in research has generated a large volume of waste mixed with scintillation liquid, requiring an effective control and appropriate storage of liquid radioactive waste. In the present study, we compared the efficacy of three commercially available scintillation liquids, Optiphase HiSafe 3, Ultima-Gold AB (biodegradable) and Insta-Gel-XF (non-biodegradable), in terms of [14C]-glucose and [ H]-thymidine counting efficiency. We also analyzed the effect of the relative amount of water (1.6 to 50%), radioisotope concentration (0.1 to 100 nCi/ml), pH (2 to 10) and color of the solutions (samples containing 0.1 to 1.0 mg/ml of Trypan blue) on the counting efficiency in the presence of these scintillation liquids. There were few significant differences in the efficiency of 14C and H counting obtained with biodegradable or non-biodegradable scintillation liquids. However, there was an 83 and 94% reduction in the efficiency of 14C and H counting, respectively, in samples colored with 1 mg/ml Trypan blue, but not with 0.1 mg/ml, independent of the scintillation liquid used. Considering the low cost of biodegradable scintillation cocktails and their efficacy, these results show that traditional hazardous scintillation fluids may be replaced with the new safe biodegradable fluids without impairment of H and 14C counting efficiency. The use of biodegradable scintillation cocktails minimizes both human and environmental exposure to hazardous solvents. In addition, some biodegradable scintillation liquids can be 40% less expensive than the traditional hazardous cocktails.

Analysis of Variance↗
PubMed2001

Effect of three different media on serum free culture of donor corneas and isolated human corneal endothelial cells.

BACKGROUND: Removal of bovine serum from organ culture medium is necessary because of the variability in serum composition and the potential risk of infection. Two specific endothelial cell media (F99 and Endothelial-SFM) were compared with the routinely used medium MEM for their use in serum free cultivation of human corneal endothelial cells (HCEC) and donor corneas. METHODS: HCEC were incubated in three test media with or without increasing serum content and a growth assay was performed. Seven pairs of donor corneas were cultured in each of three media for 3 weeks, one cornea with serum supplementation and one without. Endothelial cell density was determined once each week. Trypan blue staining of the endothelium and vital staining of keratocytes was performed after 3 weeks. RESULTS: All three media promoted proliferation of cultured HCEC when supplemented with serum. Endothelial cell density of donor corneas was comparable after 3 weeks of cultivation in the different media. Only corneas cultured in medium MEM without serum exhibited a higher endothelial cell loss. Trypan blue staining of the endothelium after cultivation revealed the lowest number of damaged cells on corneas cultured in the medium Endothelial-SFM. The highest densities of keratocytes were found in corneas cultured in Endothelial-SFM and the lowest densities occurred after culture in MEM. CONCLUSION: After incubation in Endothelial-SFM even under serum free conditions corneas were found to be of higher quality with respect to endothelial cell survival, cell membrane integrity, and keratocyte density. This medium may replace MEM, which is routinely used in European eye banks but requires supplementation with serum.

Aged↗
PubMed2003

Catha edulis (Khat) induces cell death by apoptosis in leukemia cell lines.

Khat is the Celastraceus edulis plant, a flowering evergreen tree or large shrub, which grows in the Horn of Africa and southwestern Arabia. Khat use has been associated with development of oral cancer, but its molecular effects remain controversial. This study describes a novel cytotoxic effect of whole khat extract on three leukemia cell lines. Cells were exposed to khat extract and harvested for analysis by fluorescent and electron microscopy, trypan blue exclusion, as well as immunoblotting to characterize the mode of cell death. In a separate series, cells were pretreated with a panel of caspase inhibitors for possible inhibitory effects. Khat induced a rapid cell death effect in HL-60, Jurkat, and NB4 cells that occurred within 2 h of exposure. The treated cells retained their ability to exclude trypan blue dye, a key feature in the apoptotic process. Exposed cells consistently developed morphological features of manifest apoptosis. Z-VAD, a pan-caspase inhibitor, completely inhibited toxic activity for up to 8 h, with partial inhibition by other caspase-specific agents. Western blot analysis showed specific cleavage of caspase-3 in khat-exposed cells. This study shows that khat induces cell death by apoptosis in a process sensitive to inhibition by caspase inhibitors, suggesting that subcellular interactions could be of particular relevance for the biological effects of khat in the cell death process and possibly carcinogenesis.

Apoptosis↗
PubMed2005

Epicatechin and catechin in cocoa inhibit amyloid beta protein induced apoptosis.

To elucidate additional health benefits of cocoa phytochemicals on the neurotoxicity induced by amyloid beta protein (Abeta), PC12 cells were treated with toxic peptide (Abeta(25)(-)(35)) and the effects of epicatechin, catechin, and cocoa were studied using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction, lactate dehydrogenase (LDH) release, and trypan blue exclusion methods. Significant increase in neuronal cell death was observed on PC12 cells treated with Abeta(25)(-)(35) (25 microM), while epicatechin and catechin and their mixture prevented the Abeta-induced neuronal cell death. Abeta treatment also led to the increased membrane instability of PC12 cells. The membrane protective effects of the phenolics determined by LDH release and trypan blue exclusion assays demonstrated that epicatechin, catechin, and their mixture protect cellular membrane from Abeta-induced cytotoxicity. In these three different cell viability assays, the mixture of epicatechin and catechin showed the highest protective effect and synergistic activity. The present results showed that the major flavonoids of cocoa, epicatechin and catechin, protect PC12 cells from Abeta-induced neurotoxicity, and suggest that cocoa may have anti-neurodegenerative effect in addition to other known chemopreventive effects.

Amyloid beta-Peptides↗
PubMed1997

A quinolinone derivative, vesnarinone (OPC-8212), significantly inhibits the in vitro and in vivo growth of human pancreatic cancer cell lines.

A quinolinone derivative, vesnarinone, has been used as a cardiotonic agent. Previous studies have demonstrated that vesnarinone has potent antitumor activity. The present study was designed to assess the antitumor effects of vesnarinone on human pancreatic cancer cell lines in vitro and in vivo. The in vitro effects of vesnarinone on the human pancreatic cancer cell lines (PANC-1, MIA PaCa-2 and BxPC-3) were assessed by the MTT assay, the Trypan blue dye exclusion test and the Matrigel invasion chamber assay. The inhibition of in vivo tumor growth was evaluated on two human pancreatic cancer xenografts (BxPC-3 and SPa-1) transplanted s.c. into nude mice. The dose of vesnarinone for 50% inhibition of cell growth in a 7 day culture ranged between 10 and 20 micrograms/ml as verified by the Trypan blue dye exclusion test. The dose for 50% cytotoxicity after a 3 day culture in the MTT assay was 32 micrograms/ml for PANC-1 and 30 micrograms/ml for BxPC-3, but 50% cytotoxicity for MIA PaCa-2 was not achieved by the maximal dose of vesnarinone (50 micrograms/ml). Nomalsky optic microscopy and acridine orange staining demonstrated the vacuolization and crater-like changes in the cell nucleus after vesnarinone treatment. Moreover, staining of an apoptosis marker (Le(y) protein) and nick end-labeling increased. Vesnarinone also inhibited cancer invasion in the Matrigel invasion chamber assay. In vivo, BxPC-3 and SPa-1 were s.c. transplanted into the nude mice, and vesnarinone (5 or 50 mg/kg) was daily administered orally for 21 days. In both lines, vesnarinone at 50 mg/kg achieved significant inhibition. The present study suggests that vesnarinone may be a new therapeutic agent for pancreatic cancer.

Adenocarcinoma↗
PubMed1987

Biostimulative effects of Nd:YAG Q-switch dye on normal human fibroblast cultures: study of a new chemosensitizing agent for the Nd:YAG laser.

Kodak Q-switch II is a new chemical with an absorption maxima at 1,051 nm, designed to be used as an Nd:YAG dye laser. The potential for this dye as a new chemosensitizing agent in the treatment of connective tissue diseases and wound healing with low energy Nd:YAG laser was examined. Two normal fibroblast cell lines were tested for sensitivity to various levels of this dye in vitro. These cells were exposed to Q-switch II dye at concentrations of 0.01, 0.1, 1, 10, 50, and 100 micrograms/ml for 1 and 24 hours. Cell viability was assessed by the trypan blue exclusion test. Cell duplication and DNA synthesis were measured by the incorporation of [3H]-thymidine at 6 and 24 hours postexposure to Q-switch II dye. At concentrations up to 10 micrograms/ml, both cell lines tested showed no changes in cell viability. However, at concentrations equal or higher than 50 micrograms/ml, more than 40% of the fibroblasts incorporated trypan blue after 24 hours of exposure to this dye, indicating significant cell destruction. The results indicate that Q-switch II dye is nontoxic to normal human fibroblast cultures and showed significant biostimulative effects on cell duplication at concentrations equal to or lower than 10 micrograms/ml. Further studies will be required to determine the usefulness of Q-switch II dye as a new photochemosensitizing agent for potential biostimulation of wound healing and/or treatment of connective tissue diseases with the Nd:YAG laser (near infrared, 1,060 nm) at "nonthermal" levels of energies.

Benzopyrans↗