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At least 829 records · Page 46Linked to original sources
PubMed1992

An organoid culture assay (OCA) for determining the drug sensitivity of human tumors.

A model for testing chemotherapeutic agents in vitro is described. It is based on an organoid culture method which allows human carcinomas to grow in vitro and to maintain many typical in vivo properties, including 3-dimensional architecture, growth of multiple cell types, expression of morphological differentiation and formation of histotypical structures. The preservation of drug sensitivity and resistance under the conditions of our organoid culture assay (OCA) was demonstrated by investigating 3 strains of a human hypopharynx carcinoma which differed by different sensitivity to cisplatin in in vivo conditions. These differences were retained in vitro and the modified neutral-red (NR) assay was especially suitable for revealing drug-induced cytotoxic damage in OCA. On the basis of our findings, the following approach is proposed for the in vitro testing of cytostatic drugs before they are administered to patients. (1) Removal of carcinomas from patients; (2) dense cell suspensions of these carcinomas to be dropped on membrane filters at the air-medium interface, resulting in growth of solid nodules of organized carcinoma tissues; (3) addition of cytostatic drugs to the growth medium for 2 or 3 days; (4) detachment and bisection of the culture nodules; (5) determination of viable cells by NR uptake and total cell mass by the sulforhodamin B (SRB) assay; (6) determination of quotient NR:SRB absorbance, related as percentage to control value: this indicates the fraction of viable cells and gives a measure of the cytotoxic injury caused by the applied cytotoxic drug. Thus, the OCA seems to be suitable for defining the patterns of drug sensitivity and resistance of individual human carcinomas in vitro within a few days.

Animals↗
PubMed2000

Cytotoxicity of the MEIC reference chemicals in rat hepatoma-derived Fa32 cells.

The cytotoxicity of the MEIC reference chemicals was investigated in rat hepatoma-derived Fa32 cells. The total protein content was measured as an endpoint after exposure times of 30 min and 24 h, both in normal and glutathione-depleted cells. The neutral red uptake inhibition and the MTT conversion were also measured after 30 min. On average, the cytotoxicity was higher in glutathione-depleted cells when compared to normal cells, and was lower after 30 min than after 24 h. Evidence was obtained for lysosomal attack (of five chemicals) or mitochondrial dysfunction (of six chemicals) as the primary intoxication mechanism. Malathion and mercuric chloride belong to both series of chemicals. Good to excellent correlations were observed when the 50% inhibitory concentrations of the six different in vitro assays were compared. When the six in vitro assays in Fa32 cells were compared with the human toxicity, the correlation coefficient was almost always identical to that obtained previously in human hepatoma-derived Hep G2 cells. The latter was the best acute in vitro assay for the prediction of human toxicity within the MEIC study. Altogether the results integrate very well with the basal cytotoxicity concept (Ekwall, B., 1995. The basal cytotoxicity concept. In: Goldberg, A.M., Van Zutphen, L.F.M. (Eds.), The World Congress on Alternatives and Animal Use in the Life Sciences: Education, Research, Testing. Mary Ann Liebert Publishers, New York, pp. 721-725).

Animals↗
PubMed2003

Potential antiviral therapeutics for smallpox, monkeypox and other orthopoxvirus infections.

We assessed the activities of 24 different antiviral compounds against smallpox (two strains of variola major and one of variola minor), monkeypox, vaccinia and cowpox viruses by a neutral red uptake assay. To establish assay parameters, we examined viral replication and its inhibition at various times postinfection and at several multiplicities of infection. Drugs were selected to target a range of functions involved in viral replication. Eight compounds (cidofovir, cyclic HPMPC (cHPMPC), HPMPA, ribavirin, tiazofurin, carbocyclic 3-deazaadenosine, 3-deazaneplanocin A and DFBA (1-(2,4-difluorobenzyloxy)adenosine perchlorate)-a derivative of adenosine N1-oxide) inhibited the replication of all three variola strains and the other orthopoxviruses at drug concentrations within a pharmacologically achievable range. Two others (methisazone and bis-POM-PMEA) showed a lesser degree of antiviral effect, while the remainder were inactive. To examine possible naturally occurring drug resistance among a large number of variola isolates obtained from different geographical regions and at different times, we examined the sensitivity of 35 different strains of variola as well as other orthopoxviruses to a subset of three of the most active compounds: cidofovir, cHPMPC, and ribavirin. Preliminary data indicate that nearly all isolates appear to have similar drug sensitivities. These findings are currently being verified and expanded.

Animals↗
PubMed2005

Gastroprotective activity of a new semi-synthetic solidagenone derivative in mice.

The gastroprotective activity of the new semi-synthetic solidagenone derivative 15,16-epoxy8(9),13(16),14-labdatrien-7 beta-methoxy-6 beta-ol (ELMO) has been assessed on the model of HCl/EtOH-induced gastric lesions in mice. Human gastric epithelial cells (AGS) and fibroblasts (MRC-5) were used to determine its mode of action. The effect of ELMO on the prostaglandin E2 content, cellular reduced glutathione (GSH) and protection against damage induced by sodium taurocholate was assessed against AGS cells. The effect of ELMO on the growth of AGS and fibroblast cultures was evaluated. The superoxide anion scavenging capacity of the compound was studied also. The cytotoxicity of ELMO, expressed as cell viability, was assessed using two independent endpoints: neutral red uptake (NRU) and the reduction of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) for MRC-5 fibroblasts and NRU for AGS cells. A single oral dose of ELMO (10 and 20 mg kg(-1)) inhibited the appearance of gastric lesions in mice displaying similar values to lansoprazole at 20 mg kg(-1). At 40 microM ELMO increased the prostaglandin E2 content but not GSH in AGS cells. The compound showed no effect on sodium taurocholate-induced damage and was devoid of superoxide anion scavenging activity. Concentrations of 0.5, 1, 2 and 4 microM stimulated fibroblast but not AGS cell proliferation. The compound showed weak cytotoxicity with values (IC50) of 411 (NRU) and 418 microM (MTT) for fibroblasts and 261 microM (NRU) for AGS cells. The results support further pharmacological study of this compound as a potential new anti-ulcerogenic drug.

2-Pyridinylmethylsulfinylbenzimidazoles↗
PubMed1997

Islet capillary blood pressure increase mediated by hyperglycemia in NIDDM GK rats.

This study was performed to measure pancreatic islet capillary pressure under basal conditions and after an acute glucose stimulation of insulin release in normal rats. In addition, the islet capillary pressure was estimated in GK rats, an animal model of NIDDM. Hydrostatic pressure in single pancreatic islet capillaries was determined in vivo by direct measurement using the micropuncture technique. The pancreatic islets were visualized by injection of neutral red. This intravital staining had no effect on islet function, whole pancreatic and islet blood flow, and capillary blood pressure in the exocrine pancreas. Islet capillary blood pressure in normoglycemic Wistar F rats was estimated at 3.1 +/- 0.3 mmHg (n = 15). Administration of D-glucose (1 g/kg) doubled this value, whereas no effect was seen after injection of an equimolar dose of the non-metabolizable glucose-derivative 3-O-methyl glucose. In GK rats, basal islet capillary blood pressure was increased (5.7 +/- 0.4 mmHg; n = 10; P < 0.001) when compared with the control Wistar F rats. Reduction of blood glucose levels in GK rats with phlorizin treatment showed this increased basal islet capillary pressure in GK rats to be glucose dependent and reversible. In the present study, we have for the first time shown that both acute and chronic hyperglycemia augment islet capillary pressure. The effects of a chronically increased islet capillary pressure on long-term islet function remain to be determined.

3-O-Methylglucose↗
PubMed2003

Antioxidants protect primary rat hepatocyte cultures against acetaminophen-induced DNA strand breaks but not against acetaminophen-induced cytotoxicity.

Acetaminophen, a safe analgesic when dosed properly but hepatotoxic at overdoses, has been reported to induce DNA strand breaks but it is unclear whether this event preceeds hepatocyte toxicity or is only obvious in case of overt cytotoxicity. Moreover, it is not known whether the formation of reactive oxygen species (ROS) is involved in the formation of the DNA strand breaks. In the present study, the dose-response curves for cytotoxicity and DNA strand breaks and the response to antioxidant protection have been compared. In primary hepatocytes from untreated male rats, cytotoxicity as measured by the MTT test and by Neutral Red accumulation was obvious at 10 mM acetaminophen but DNA strand breaks as measured by the comet assay were only found at 25-30 mM acetaminophen. Non-cytotoxic concentrations of three compounds with antioxidant activity, the glutathione precursor N-acetylcysteine (100 micro M), the plant polyphenol silibin (25 micro M) and the antioxidant vitamin alpha-tocopherol (50 micro M), were not able to inhibit acetaminophen toxicity at any acetaminophen concentration, while they completely prevented the formation of DNA strand breaks at 25-30 mM acetaminophen. The occurrence of oxidative stress in our experiments was indicated by a slight increase of malondialdehyde formation at 40 mM acetaminophen and by an adaptive increase in catalase mRNA concentration. We conclude that in acetaminophen-treated hepatocytes ROS-independent cell death and ROS-dependent DNA strand breaks occur which appear not to be causally related as judged from their dose dependency and their response to antioxidants.

Acetaminophen↗
PubMed1994

Selection of cytotoxic responses to maitotoxin and okadaic acid and evaluation of toxicity of dinoflagellate extracts.

The cytotoxicity of maitotoxin (MTX) and okadaic acid (OA) was studied on three mammalian fibroblast cell lines. Neutral red uptake (NRU), which measures cell viability, and morphological alterations were selected as rapid suitable responses. NRU allowed a precise toxicity quantification while the observations of morphological damage revealed differences specific to MTX (cell blebbing) and OA (cell rounding). BHK21 C13 fibroblasts, although less sensitive to MTX than the other cell lines, were chosen since they gave stable information and a two-stage morphological response with OA ("square"-shaped cells, then round cells). When NRU and morphology alterations were studied with crude extracts of Gambierdiscus toxicus and Prorocentrum lima, responses were typical of the dominant toxins, MTX and OA or related toxins respectively. Applied to several dinoflagellate extracts, the two tests revealed no toxicity for Amphidinium carterae, Ostreopsis siamensis, O. ovata and Coolia monotis (from La Réunion) and toxicity for A. carterae and A. operculatum (from Saint Barthélémy). When toxic, A. carterae extracts showed blebbing similar to that caused by MTX. Morphology alterations caused by A. operculatum crude extracts, different from those corresponding to MTX or OA, were also observed.

3T3 Cells↗
PubMed2002

Preliminary evaluation of the cytotoxic and genotoxic potential of D-003: Mixture of very long chain fatty acids.

D-003 is a mixture of very long chain aliphatic acids purified from sugar cane wax, wherein octacosanoic acid represents the major component. Previous experimental studies have shown that D-003 inhibits platelet aggregation in rodents. Also, its lowers total (TC) and low-density lipoprotein cholesterol (LDL-C) in normocholesterolemic rabbits in a dose-dependent manner and inhibits cholesterol biosynthesis in fibroblast cultures. The present study was performed to investigate the in vitro cytotoxic and genotoxic potential effects of D-003 assessed through two tests: the neutral red (NR) assay and the Ames test. Positive and negative controls were included in each experimental series. Compared with controls, no cytotoxicity was evident after 24 and 72 h of treatment with doses up to 1,000 microg/ml in the NR assay. On the other hand, D-003 (5-5,000 microg/plate) did not increase the frequency of reverse mutations in the Ames test in both alternatives with or without S9 mix metabolic activation and a pre-incubation step. The positive control chemicals included in each experiment, namely, treatment with sodium dodecyl sulphate (SDS) in the NR assay and sodium azide (NaAz), 2-aminofluorene (AF), and dimethylnitrosamine (DMNA) in the Ames test, induced the expected changes, such as a decrease in optical density (OD) values in the NR assay and an increase in the frequency of reverse mutations in the Ames test. The present results indicate that D-003 did not show evidence of cytotoxic or genotoxic potential in tests able to detect the ability of chemicals to disrupt cells (NR assay) or to induce gene mutations (Ames test).

3T3 Cells↗
PubMed2000

Hemocytic lysosome response in the snail Helix aspersa after exposure to the fungicide copper oxychloride.

This study investigated the use of lysosomal responses of hemocytes of the common garden snail, Helix aspersa, as biomarker of stress due to exposure to the fungicide copper oxychloride. The neutral red retention (NRR) time assay was employed for this purpose. Two groups of snails were exposed to 80 microg g(-1) and 240 microg g(-1) copper oxychloride in their food, respectively, for a period of 6 weeks. They were compared with a control group to which no copper oxychloride was added. The two groups exposed to the fungicide exhibited significantly higher (p < 0.001) whole body copper concentrations (200.85 +/- 53.5 and 272.24 +/- 67.15 microg g(-1) dry mass, respectively), and significantly shorter (p < 0.001) NRR times (10.22 +/- 3.53 and 2.67 +/- 2.83 min, respectively), after 6 weeks, compared to the control group (67.85 +/- 31.08 microg g(-1) dry mass and 24.44 +/- 8.35 min). In both exposure groups NRR times became progressively shorter as body copper concentrations increased over time. Thus, both exposure concentration and exposure time of copper oxychloride were shown to be important factors influencing lysosomal responses (and therefore NRR times) of H. aspersa hemocytes. It was concluded that these responses in this species, as measured by the NRR time assay, could be considered a useful cellular biomarker of stress resulting from exposure to copper oxychloride.

Animals↗
PubMed1998

Intracellular alkalinisation in Vero cells parasitised by Trypanosoma cruzi.

We studied the intracellular pH of Vero cells parasitised by Trypanosoma cruzi, using different methods: fluorimetric measurement after labelling the cells with the pH-sensitive intracellular fluorescent dye 2',7',-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester; flow cytometry; and image analysis after staining the cells with neutral-red vital stain. The results show that the intracellular pH of the parasitised cells rose in comparison with that of the uninfected control cells. A study of the population of parasitised cells made by flow cytometry allowed us to subdivide the cells from the infected cultures into two populations according to their pH as obtained by fluorimetric measurements. Image analysis showed that the cell cytoplasm was more alkaline in the vicinity of the sites containing parasites. Treatment of the parasitised cells with amiloride, ouabain, or with 4.4'-diisothiocyano-2,2'-stilbene disulphate consistently lowered the pH values of the parasitised cells, but not sufficiently to return to the values of the non-parasitised control cells. When the control cells were subject to similar treatments with the inhibitors, only amiloride acidified the cytoplasm to any extent. The basification undergone by the parasitised cells was independent of the transport systems and may be a consequence of the release of NH4+ by the intracellular amastigotes.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1990

Gravity-dependent polarity of cytoplasmic streaming in Nitellopsis.

The internodal cells of the characean alga Nitellopsis obtusa were chosen to investigate the effect of gravity on cytoplasmic streaming. Horizontal cells exhibit streaming with equal velocities in both directions, whereas in vertically oriented cells, the downward-streaming cytoplasm flows ca. 10% faster than the upward-streaming cytoplasm. These results are independent of the orientation of the morphological top and bottom of the cell. We define the ratio of the velocity of the downward- to the upward-streaming cytoplasm as the polar ratio (PR). The normal polarity of a cell can be reversed (PR < 1) by treatment with neutral red (NR). The NR effect may be the result of membrane hyperpolarization, caused by the opening of K+ channels. The K+ channel blocker TEA Cl- inhibits the NR effect. External Ca2+ is required for normal graviresponsiveness. The [Ca2+] of the medium determines the polarity of cytoplasmic streaming. Less than 1 micromole Ca2+ resulted in a PR < 1 while greater than 1 micromole Ca2+ resulted in the normal gravity response. The voltage-dependent Ca(2+)-channel blocker, nifedipine, inhibited the gravity response in a reversible manner, while treatment with LaCl3 resulted in a PR < 1, indicating the presence of two types of Ca2+ channels. A new model for graviperception is presented in which the whole cell acts as the gravity sensor, and the plasma membrane acts as the gravireceptor. This is supported by ligation and UV irradiation experiments which indicate that the membranes at both ends of the cell are required for graviperception. The density of the external medium also affects the PR of Nitellopsis. Calculations are presented that indicate that the weight of the protoplasm may provide enough potential energy to open ion channels.

Calcium↗
PubMed1992

Quantitative measurement of two-component pH-sensitive colorimetric spectra using multilayer neural networks.

The purpose of this research was to develop a noise tolerant and faster processing approach for in vivo and in vitro spectrophotometric applications where distorted spectra are difficult to interpret quantitatively. A PC based multilayer neural network with a sigmoid activation function and a generalized delta learning rule was trained with a two component (protonated and unprotonated form) pH-dependent spectrum generated from microspectrophotometry of the vital dye neutral red (NR). The network makes use of the digitized absorption spectrum between 375 and 675 nm. The number of nodes in the input layer was determined by the required resolution. The number of output nodes determined the step size of the quantization value used to distinguish the input spectra (i.e. defined the number of distinct output steps). Mathematic analysis provided the conditions for which this network is guaranteed to converge. Simulation results showed that features of the input spectrum were successfully identified and stored in the weight matrix of the input and hidden layers. After convergent training with typical spectra, a calibration curve was constructed to interpret the output layer activity and therefore, predict interpolated pH values of unknown spectra. With its built-in redundant presentation, this approach needed no preprocessing procedures (baseline correction or intensive signal averaging) normally used in multicomponent analyses. The identification of unknown spectra with the activities of the output layer is a one step process using the convergent weight matrix. After learning from examples, real time applications can be accomplished without solving multiple linear equations as in the multiple linear regression method.(ABSTRACT TRUNCATED AT 250 WORDS)

Hydrogen-Ion Concentration↗
PubMed1996

Apoptosis in human monocyte-macrophages exposed to oxidized low density lipoprotein.

This study has demonstrated the toxicity to human monocyte-macrophages of low-density lipoprotein (LDL) which had been artificially oxidized using copper sulphate. The assays of cell damage used were tritiated adenine release, neutral red staining, lactate dehydrogenase leakage, and MTT dye reduction. Toxicity was concentration- and time-dependent. Exposure to native LDL under the same conditions did not result in toxicity. Transmission electron microscopy of cells exposed to oxidized LDL showed characteristic changes of apoptosis, including chromatin condensation and a decrease in cell volume. There was extensive loss of cell surface protrusions and evidence of the phagocytosis of apoptotic cells by neighbouring monocyte-macrophages. Apoptotic features preceded the increased membrane permeability revealed by the release of radioactivity from cells preloaded with tritiated adenine and by lactate dehydrogenase leakage. DNA fragmentation was indicated by nick end-labelling using the terminal transferase enzyme (TUNEL). The number of TUNEL-positive cells was markedly greater in cells exposed to oxidized LDL, compared with those incubated as no-additions controls. Inhibition of de novo protein synthesis with cycloheximide and of Ca2+/Mg(2+)-activated endonuclease activity with aurintricarboxylic acid or zinc ion did not inhibit the toxicity produced by oxidized LDL.

Adenine↗
PubMed1997

In vitro cytotoxic effects of 4,4'-bipyridyl on normal human keratinocytes.

Recent epidemiological studies have brought to light a possible link between premalignant or neoplastic skin lesions (Bowen disease, squamous carcinoma) and occupational exposure to 4,4'-bipyridyl (4,4'B), a precursor in the synthesis of paraquat herbicide. The present study used a serum-free cell culture of normal human keratinocytes (NHK) and two skin-equivalent models to test the effects of exposure to different concentrations of 4,4'B. Cytotoxicity of 4,4'B on NHK was measured by neutral red release assay. Superoxide dismutase (SOD) activity and cell cycle were analyzed in exposed and nonexposed NHK cultures. Histological and immunohistological tests enabled evaluation of differentiation and proliferation effects in reconstructed-skin models. Results showed that significant cytotoxicity occurred after 5 to 11 days' exposure to 4,4'B concentrations of 10(-6)-10(-3) mol/L (IC50 between 10(-3) and 10(-4) mol/L 4,4'B after 11 days). Parallel modifications of SOD activity were recorded. Histological and immunohistological analysis revealed dose-related 4,4'B effects in reconstructed skin models. This involved abnormal terminal differentiation, connected with filaggrin expression, observed in skin models exposed to 10(-7) and 10(-6) mol/L 4,4'B. However, no modification of cell cycle or dysplasia was detected as a result of exposure to 4,4'B. Thus, 4,4'B appears to be cytotoxic for NHK, but as an isolated contaminant, and is unable to induce keratinocyte dysplasia in vitro. These preliminary results do not exclude a cocarcinogenic action of 4,4'B (with UVB for example).

Cell Cycle↗
PubMed2005

Genotoxicity of inorganic lead salts and disturbance of microtubule function.

Lead compounds are known genotoxicants, principally affecting the integrity of chromosomes. Lead chloride and lead acetate induced concentration-dependent increases in micronucleus frequency in V79 cells, starting at 1.1 microM lead chloride and 0.05 microM lead acetate. The difference between the lead salts, which was expected based on their relative abilities to form complex acetato-cations, was confirmed in an independent experiment. CREST analyses of the micronuclei verified that lead chloride and acetate were predominantly aneugenic (CREST-positive response), which was consistent with the morphology of the micronuclei (larger micronuclei, compared with micronuclei induced by a clastogenic mechanism). The effects of high concentrations of lead salts on the microtubule network of V79 cells were also examined using immunofluorescence staining. The dose effects of these responses were consistent with the cytotoxicity of lead(II), as visualized in the neutral-red uptake assay. In a cell-free system, 20-60 microM lead salts inhibited tubulin assembly dose-dependently. The no-observed-effect concentration of lead(II) in this assay was 10 microM. This inhibitory effect was interpreted as a shift of the assembly/disassembly steady-state toward disassembly, e.g., by reducing the concentration of assembly-competent tubulin dimers. The effects of lead salts on microtubule-associated motor-protein functions were studied using a kinesin-gliding assay that mimics intracellular transport processes in vitro by quantifying the movement of paclitaxel-stabilized microtubules across a kinesin-coated glass surface. There was a dose-dependent effect of lead nitrate on microtubule motility. Lead nitrate affected the gliding velocities of microtubules starting at concentrations above 10 microM and reached half-maximal inhibition of motility at about 50 microM. The processes reported here point to relevant interactions of lead with tubulin and kinesin at low dose levels.

Animals↗
PubMed1994

Cytotoxic effects of pamidronate on monolayers of human intestinal epithelial (Caco-2) cells and its epithelial transport.

Pamidronate (APD) is a new drug in the treatment of osteolytic bone diseases. Caco-2 cells were used to study the cytotoxic effects of APD on intestinal epithelium and also the transport (mechanism) of APD across the intestinal epithelium. We investigated the cytotoxic effect of APD by combining two spectrophotometric assays [neutral red (NR) uptake and lactate dehydrogenase (LDH) release] with a morphological assay (electron microscopy). The amount of APD transported across the Caco-2 monolayer was measured by HPLC. The permeability of the monolayer was studied by determining the transepithelial electrical resistance (TEER). The results show that after exposing the Caco-2 cells to increasing concentrations of APD [dose range calculated on the basis of relevance to the oral dose administered to patients] the NR uptake decreased while LDH loss increased, which is indicative of a cytotoxic effect of APD. Ultrastructural alterations, including a widening in intercellular spaces and, at higher doses, complete cell death, were observed. The transport percentage of nontoxic doses of APD was low, while the TEER decreased with increasing doses of APD. In conclusion, APD is cytotoxic for Caco-2 cells. As the transport percentage of nontoxic doses of APD is low and APD reduces the TEER, it is hypothesized that APD is transported paracellularly.

Biological Transport↗
PubMed2000

Photocytotoxicity of hypericin in normoxic and hypoxic conditions.

The normoxic and hypoxic photocytotoxicity of hypericin has been examined on A431 cells as assessed by the Neutral Red method, using cell-culture flasks made of polystyrene and glass, different hypericin concentrations and light fluences. Using polystyrene flasks, lower hypoxic photoactivities of hypericin than those in normoxic conditions are seen under low fluence. In these conditions the hypoxic photocytotoxic effect can be (partially) rescued by increasing the fluence. However, a completely different outcome is observed when using glass flasks, since most of the hypoxic photocytotoxicity is lost under these conditions. The differences can be explained in terms of efficiency of deoxygenation of the medium present in polystyrene or glass flasks. Polystyrene holds large amounts of oxygen that effuses very slowly. Glass, on the other hand, does not cause this inconvenience. Therefore the type of material of the container used to investigate the oxygen dependency of the photobiological activity of photosensitizers dramatically influences the outcome of the hypoxic experiments. Our results unequivocally prove that the cytotoxic effect induced by photoactivated hypericin is completely oxygen dependent. Hence hypericin does not differ from other phototherapeutics used in photodynamic therapy of cancer, since haematoporphyrin derivative and the second-generation photosensitizers used all seem to depend on the presence of oxygen for their antitumour activity.

Anthracenes↗
PubMed2001

Membrane orientation of droplets prepared from Chara corallina internodal cells.

It is generally accepted that the membrane surrounding droplets from characean cells originates from the tonoplast, but there is some uncertainty regarding droplet membrane sidedness. This issue was addressed directly by combining two different droplet isolation methods and the patch clamp technique. Neutral red accumulation was used to demonstrate the presence of H(+)-transport over the membrane and to predict membrane orientation. Two types of droplet populations with differently oriented membranes could be formed in an iso-osmotic bath solution. Cytoplasmic droplets (cytosolic side of the tonoplast inside) contained cytoplasm, while the second type of droplet population contained vacuolar sap (vacuolar droplets, vacuolar side of the tonoplast inside). Smaller vesicels also appeared inside the droplets, with an apparently inversely oriented membrane. Confocal laser scanning microscopy indirectly demonstrated that, at least with one of the droplet isolation methods, the plasma membrane entirely remains in the internodal cell after intracellular perfusion. Both types of droplet populations allowed the formation of excised patches and single-channel measurements by the patch clamp technique. Properties of anion channels in the tonoplast could be used to prove the predicted membrane orientation, knowing that Ca2+ can only activate these channels from the cytosolic side. These results provide useful data for studies addressing ligand-binding, block and modulation, organization and interaction of proteins within the membrane or with other regulatory factors, where it is important to control membrane orientation.

Cell Membrane↗