The Antioxidant Effect of Fermented Papaya Preparation in the Oral Cavity.

Oxidative stress has been recognized to play important roles in various diseases, including of the oral cavity. However, nutritional supplementation of antioxidants to ameliorate the consequences of oxidative stress is debatable. One caveat is that oxidative status is often measured under non-physiological conditions. Here, we investigated the antioxidant potential of fermented papaya preparation (FPP), a product of yeast fermentation of Carica papaya Linn, under conditions that prevail in the oral cavity. Employing highly sensitive luminol-dependent chemiluminescence assays, we show that its antioxidant capacity was augmented by saliva (up to 20-fold, p < 0.0001, at 10 mg) and its components (mucin, albumin) as well as by red blood cells (RBC) and microorganisms present in the normal and pathological environment of the oral cavity. Polyphenols are major plant antioxidants. Using the Folin-Ciocalteu's assay, a very low amount of phenols was measured in FPP suspended in a salt solution. However, its suspension in saliva, albumin, mucin or RBC produced up to sixfold increase, p < 0.001, compared with the sum of polyphenols assayed separately. The results suggested that these enhancing effects were due to the solubilization of antioxidant polyphenols in FPP by saliva proteins and the binding to RBC and microorganisms, thus increasing their availability and activity. Copyright © 2015 John Wiley & Sons, Ltd.

The antioxidant effect of fermented papaya preparation involves iron chelation.

Iron-overload is a major clinical problem in various diseases. Under this condition, serum iron which surpasses the binding capacity of transferrin is present as non-transferrin bound iron and cellular unbound Labile Iron Pool (LIP) is increased. LIP participates in the generation of free radicals, including reactive oxygen species (ROS). Increased ROS, with concomitant decrease in anti-oxidants, results in oxidative stress and toxicity to the liver, heart and other tissues, causing serious morbidity and eventually mortality. Therapeutic iron chelation reduces the LIP and thereby ameliorates oxidative stress-mediated toxicity. Many food-derived antioxidants have the capacities to scavenge ROS and chelate iron. We have reported that fermented papaya preparation (FPP) has ROS scavenging effect on blood cells in vitro or in vivo (in thalassemic patients and experimental animals). We now investigated FPP's iron chelating effect - its ability to prevent (and revert) LIP accumulation. Liver- and heart-derived cells, and RBCs were exposed to non-transferrin bound iron in the form of ferrous ammonium sulfate and the effect of FPP on their LIP content and ROS generation was measured by flow-cytometry. The results indicate that FPP reduces LIP and ROS, and suggests that its antioxidant mechanism is related, at least in part, to iron chelation.

Polycations and polyanions in SARS-CoV-2 infection.

We hypothesize that polycations, such as nuclear histones, released by neutrophils COVID-19 aggravate COVID-19 by multiple mechanisms: (A) Neutralization of the electrostatic repulsion between the virus particles and the cell membrane, thereby enhancing receptor-mediated entry. (B) Binding to the virus particles, thereby inducing opsonin-mediated endocytosis. (C) Adding to the cytotoxicity, in conjunction with oxidants, cytokines and other pro-inflammatory substances secreted by cells of the innate immunity system. These effects may be alleviated by the administration of negatively charged polyanions such as heparins and heparinoids.

Tumor promoters inhibit morphological differentiation in cultured mouse neuroblastoma cells.

When added to mouse neuroblastoma cultures, the potent tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) inhibits spontaneous neurite formation as well as that induced in response to serum deprivation, prostaglandin E1, 5-bromo-2'-deoxyuridine, and papaverine. Other tumor-promoting macrocyclic plant diterpenes also inhibit neurite formation, whereas nonpromoting diterpenes do not. Inhibition by TPA was reversible and was unrelated to toxicity.

Growth hormone and erythropoietin differentially activate DNA-binding proteins by tyrosine phosphorylation.

Binding of growth hormone (GH) and erythropoietin (EPO) to their respective receptors results in receptor clustering and activation of tyrosine kinases that initiate a cascade of events resulting not only in the rapid tyrosine phosphorylation of several proteins but also in the induction of early-response genes. In this report, we show that GH and EPO induce the tyrosine phosphorylation of cellular proteins with molecular masses of 93 kDa and of 91 and 84 kDa, respectively, and that these proteins form DNA-binding complexes which recognize an enhancer that has features in common with several rapidly induced genes such as c-fos. Assembly of the protein complexes required tyrosine phosphorylation, which occurred within minutes after addition of ligand. The activated complexes translocated from the cytoplasm to the nucleus. The protein activated by GH is antigenically similar to p91, a protein common to several transcription complexes that are activated by interferons and other cytokines. In contrast, the proteins activated by EPO are distinct from p91. These findings establish the outlines for a cytokine-induced intracellular signaling pathway, which begins with ligand-induced receptor clustering that activates one or more tyrosine kinases. These data are the first to demonstrate that GH- and EPO-activated tyrosine-phosphorylated proteins can specifically recognize a well-defined enhancer and therefore provide a mechanism for rapidly transducing signals from the membrane to the nucleus.

The antiepileptic and anticancer agent, valproic acid, induces P-glycoprotein in human tumour cell lines and in rat liver.

BACKGROUND AND PURPOSE: The antiepileptic drug valproic acid, a histone deacetylase (HDAC) inhibitor, is currently being tested as an anticancer agent. However, HDAC inhibitors may interact with anticancer drugs through induction of P-glycoprotein (P-gp, MDR1) expression. In this study we assessed whether valproic acid induces P-gp function in tumour cells. We also investigated effects of valproic acid on the mRNA for P-gp and the cytochrome P450, CYP3A, in rat livers. EXPERIMENTAL APPROACH: Effects of valproic acid on P-gp were assessed in three tumour cell lines, SW620, KG1a and H4IIE. Accumulation of acetylated histone H3 in rats' livers treated for two or seven days with valproic acid was evaluated using a specific antibody. Hepatic expression of the P-gp genes, mdr1a, mdr1b and mdr2, was determined by real-time polymerase chain reaction. The effects of valproic acid on CYP3A were assessed by Northern blot analysis and CYP3A activity assays. KEY RESULTS: Valproic acid (0.5-2.0 mM) induced P-gp expression and function up to 4-fold in vitro. The effect of a series of valproic acid derivatives on P-gp expression in SW620 and KG1a cells correlated with their HDAC inhibition potencies. Treatment of rats with 1 mmol kg(-1) valproic acid for two and seven days increased hepatic histone acetylation (1.3- and 3.5-fold, respectively) and the expression of mdr1a and mdr2 (2.2-4.1-fold). Valpromide (0.5-2.0 mM) did not increase histone acetylation or P-gp expression in rat livers, but induced CYP3A expression. CONCLUSIONS: Valproic acid increased P-gp expression and function in human tumour cell lines and in rat liver. The clinical significance of this increase merits further investigation.

Spontaneous commitment of murine erythroleukemic cells to terminal differentiation.

Differentiation in murine erythroleukemic cells is arrested at the proerythroblast stage. A small fraction of the population, however, undergoes spontaneous differentiation. This spontaneous differentiation was examined at the individual cell level in relation to cell multiplication, commitment, and maturation. The results indicate that murine erythroleukemic cells destined to undergo spontaneous differentiation first undergo commitment, an irreversible process characterized by cells becoming (a) capable of producing hemoglobin coupled with (b) a loss of their ability to undergo more than six subsequent cell divisions. Commitment is followed by a maturation process which includes the accumulation of erythroid specific markers, e.g., hemoglobin. With respect to commitment, spontaneous differentiation resembles the differentiation produced by most inducers but differs from that evoked by hemin. Serum hemin may therefore be exempt from implication in the spontaneous process. 12-O-Tetradecanoylphorbol-13-acetate, which is known to inhibit murine erythroleukemic cell differentiation, was found to exert its inhibitory effect on both the commitment and maturation steps of spontaneous differentiation. The results further indicate that cells become committed mainly during the logarithmic rather than the stationary phase of the growth cycle. Once committed, however, the cells mature during both the logarithmic and stationary phases. When logarithmic growth was maintained continuously, the rate of the spontaneous differentiation increased (20- to 100-fold) due to the higher probability of cell commitment. A steady state culture was obtained in which the rates of cell multiplication, initiation of commitment, and maturation remained constant.

Changes in DNA associated with induction of erythroid differentiation by dimethyl sulfoxide in murine erythroleukemia cells.

The Friend virus-infected murine erythroleukemia cell can be induced to differentiate along erythroid cells in culture with various compounds, including dimethyl sulfoxide. DNA from murine erythroleukemia cells cultured with dimethyl sulfoxide shows a decrease in sedimentation rate in alkaline sucrose gradients after alkali lysis of the cells. These changes can be detected as early as 27 hr after the beginning of culture. Similar results are observed with DNA of the cells cultured with other inducers, butyric acid and dimethylacetamide, but not with DNA from a variant cell line resistant to induction with dimethyl sulfoxide. Ultraviolet irradiation, which is known to cause similar changes in the sedimentation rate of DNA in alkaline sucrose gradients, induces differentiation of the murine erythroleukemia cells. These studies suggest that alterations in DNA may be related to events involved in the induction of differentiation of murine erythroleukemia cells by dimethyl sulfoxide.

Effect of hexamethylene bisacetamide on the commitment to differentiation of murine erythroleukemia cells.

Friend virus-transformed murine erythroleukemia cells express the program of erythropoietic differentiation under the influence of the previously described, potent inducing agent, hexamethylene bisacetamide. Commitment to differentiation, defined as the ability to continue the processes of differentiation in the absence of inducer, has been examined at the single-cell level, with a combination of suspension and cell-cloning techniques. Recruitment of committed cells is shown to occur prior to the detectable accumulation of hemoglobin or the appearance of morphological changes characteristic or erythroid maturation. The stability of the commitment of murine erythroleukemia cells to differentiate is found to be dependent upon both the concentration of hexamethylene bisacetamide and the duration of exposure to the inducing agent. Under conditions less than optimal for induction, a single cell can give rise to a colony containing both differentiated and undifferentiated cells. On the basis of these findings, it is suggested that fully stabilized differentiation, in addition to the previously demonstrated requirement for the inducing agent to be present during a cell-cycle S phase, involves subsequent stabilizing event(s) caused by a direct or indirect action of the inducing agent.

Heterogeneity of murine erythroleukemia cells with respect to tumor promoter-mediated inhibition of cell differentiation.

Spontaneous and induced differentiation of murine erythroleukemia cells (strain 745A DS19 ) is reversibly inhibited by 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent promoter of mouse skin carcinogenesis, and by other tumor-promoting macrocyclic plant diterpenes, but it is not by nonpromoting diterpenes. Twelve clones randomly isolated from this strain vary in their response to TPA. All clones are induced to differentiate by several compounds, the most potent of which is hexamethylene bisacetamide. In six clones TPA (100 ng/ml) caused greater than 90% inhibition of differentiation, as measured by the appearance of benzidine-reactive cells. In two clones cell differentiation was not inhibited by TPA even at concentrations as high as 1 microgram/ml. In four clones, differentiation was only partially inhibited (16 to 47%) by TPA. Clones resistant to TPA inhibition of differentiation were also resistant to structurally related tumor-promoting agents. The isolation of variant cell lines, sensitive and resistant to TPA, provides a tool for elucidating the mechanism of tumor promoter-mediated inhibition of cell differentiation.

Human leukemic myeloblasts and myeloblastoid cells contain the enzyme cytidine 5'-monophosphate-N-acetylneuraminic acid:Gal beta 1-3GalNAc alpha (2-3)-sialyltransferase.

We have examined the role of CMP-NeuAc:Gal beta 1-3GalNAc-R alpha(2-3)-sialyltransferase in fresh leukemia cells and leukemia-derived cell lines. Enzyme activity in normal granulocytes using Gal beta 1-3GalNAc alpha-o-nitrophenyl as substrate was 1.5 +/- 0.7 nmol/mg/h whereas activity in morphologically mature granulocytes from 6 patients with chronic myelogenous leukemia (CML) was 4.2 +/- 1.6 nmol/mg/h (P less than 0.05). Myeloblasts from 5 patients with CML in blast crisis showed enzyme activity levels of 6.5 +/- 2.5 nmol/mg/h. From 2 patients with CML, both blasts and granulocytes were obtained, with higher enzyme activity in the patients' blasts (7.1 nmol/mg/h) than in their granulocytes (4.9 nmol/mg/h) in both cases, suggesting that the increase in enzyme activity is related to the differentiation or proliferation status of the CML cells. However, similarly high enzyme levels were also seen in myeloblasts from acute myeloblastic leukemia patients (5.6 +/- 1.4 nmol/mg/h) and in some acute myeloblastic leukemia-derived cell lines (KG1a and HL60), suggesting that increased levels of this enzyme are not directly correlated with the presence of the Ph1 chromosome. This alpha(2-3)-sialyltransferase activity can also be detected in normal peripheral blood lymphocytes and exhibits increased activity in chronic lymphocytic leukemia cells and acute lymphoblastic leukemia. These data suggest that the level of enzyme activity may vary with growth rate and maturation status in myeloid and lymphoid hemopoietic cells. Finally, we have identified a glycoprotein in acute myeloblastic leukemia cells that serves as a substrate for the alpha(2-3)-sialyltransferase. The desialylated form of the glycoprotein was resialylated in vitro by the purified placental form of this alpha(2-3)-sialyltransferase and exhibits a molecular weight of about 150,000.

Spectrofluorometric measurements of the dispersion state of pyrenedodecanoic acid and its uptake by cultured cells and liposomes.

Pyrene dodecanoic acid (P12), a medium-chain fatty acid to which the fluorescent probe pyrene is covalently linked, showed a considerable increase in fluorescence when the probe was introduced into a hydrophobic environment. Also, when closely packed in an aggregate, an energy transfer between two adjacent molecules of pyrene occurred, resulting in a shift of the peak of the emission spectrum from 378 nm ('monomeric') to 475 nm ('excimeric'). These two respective properties were utilized for the following: (a) A spectrofluorometric measurement of the critical micellar concentration (CMC) of the pyrene fatty acid, defined as the concentration at which the 475 nm emission peak appeared as a consequence of the aggregation of P12 molecules in aqueous solution to form micelles; the CMC of P12 was found to be in the range of 1 to 2 microM. (b) The penetration of P12, from an aqueous solution or dispersion, into unilamellar phospholipid vesicles was determined by monitoring the increase of the fluorescence at 378 nm. The fluorescence increase was time-dependent and proportional to the respective concentrations of P12 or phospholipid vesicles. Substituting the neutral phosphatidylcholine with the negatively-charged phosphatidylserine vesicles resulted in a slower rate as well as lesser total uptake of P12. (c) The uptake of P12 by cells was accompanied by an increase in the monomeric fluorescence emission intensity. Using cells in suspension, this could be followed continuously in a spectrofluorometer equipped with a recorder. The uptake was found to be time-dependent and proportional to P12 concentration.

Effect of albumin, low temperature and metabolic inhibitors on transport of fatty acids into cultured human leukemic myeloid cells.

Radioactively-labelled palmitic acid was used to study the effects of albumin, low temperature and several inhibitors of metabolism on transport of fatty acids into cultured human leukemic myeloid cells. When serum or albumin were present in the medium, uptake of fatty acid by cells as well as its further incorporation into phospholipids and neutral lipids were considerably reduced. Uptake and metabolic utilization of this fatty acid was reduced at low temperature, in the presence or absence of albumin in the incubation medium. In absence of albumin, addition of iodoacetate, sodium cyanide or sodium azide had but little effect on the total uptake of fatty acids while metabolic utilization was reduced. When albumin was present, these inhibitors reduced both total uptake and incorporation into lipids. The data suggest that incorporation of the fatty acid into the outer layer of the cell membrane is controlled by the concentration of free, uncomplexed molecules of fatty acid adjacent to the cell surface. In the absence of albumin this is a fast reaction which reaches nearly maximal uptake in three minutes. In the presence of albumin, this process is much slower and follows a nearly linear course between 3 and 60 minutes. Translocation into the inner layer of the membrane and subsequent utilization for metabolic processes is a much slower process, which seems to depend on the quantity of the fatty acid in the outer layer.

Use of the fluorescence activated cell sorter for studying uptake of fluorescent fatty acids into cultured cells.

A procedure is described for the continuous monitoring and recording of fatty acid transport into cultured cells. Uptake of the fluorescent fatty acid derivative, 12-(1-pyrene)dodecanoic acid [P12] by a suspension of human promyelocytic leukemia (HL-60) cells was analyzed and recorded by the Fluorescence Activated Cell Sorter (FACS). A major advantage of the FACS is its ability for measuring the fluorescence of the cells only, without interference by that of the fatty acids dissolved or dispersed in the medium or complexed with serum albumin. The time-dependent fluorescence increase due to uptake of the acid by the cells could thus be monitored and recorded directly without a need for sedimenting, washing and extracting the cellular lipids. This procedure provided an accurate measurement of the kinetics of uptake of the fatty acid into the cells and permitted study of the effect of various parameters such as temperature, glucose, albumin or serum. The results indicated a biphasic uptake of P12 into the cells. The first, a rapid, energy-independent phase, lasted 3-4 min. This was followed by a slower uptake which was directly related to the metabolic utilization of the acid in the cells. This second phase represents the overall process of translocation across the cell membrane, activation to acyl coenzyme A and incorporation into the cellular neutral lipids and phosphoglycerides. When compared to HL-60, murine erythroleukemia (MEL) cells took up considerably lesser quantities of P12. This was utilized for setting up a model system, in which mixtures of these two respective cell types could be identified and separated from each other on the basis of their relative rates of uptake of the fluorescent fatty acid.

Induction of lipid storage in cultured leukemic myeloid cells by pyrene-dodecanoic acid.

When incubated for 1-3 days in the presence of the fatty acid analog, 12-(1-pyrene)dodecanoic acid, the neutral lipid content of cultured human leukemic myeloid cells increased considerably, while that of the phospholipids increased to a much lesser extent. Among the neutral lipids, di- and monoacylglycerols predominated and a considerable portion of the fatty acyl residues of these newly synthesized neutral lipids consisted of pyrene-dodecanoic acid. Light microscopy showed evidence for the presence of highly fluorescent lipid droplets within the cells. Electron microscopy showed lipid globules, mostly devoid of a unit membrane, multivesicular inclusion bodies and some multilamellar membranous structures. In comparison, cells incubated with palmitic acid show neither these cellular structures, nor the increase of the neutral lipid content. The lipid storage, induced by pyrene-dodecanoic acid, is probably related to ineffective degradation of this fatty acid analog and might serve as an experimental model of cellular lipidosis.

Transport of fluorescent derivatives of fatty acids into cultured human leukemic myeloid cells and their subsequent metabolic utilization.

Transport of fluorescent derivatives of fatty acids across the cell membrane of cultured human leukemic myeloid cells (HL 60) and their subsequent metabolic utilization were studied. The rates of uptake of these derivatives and their incorporation into cellular lipids wer compared with that of radioactively labelled palmitic acid. Three groups of fluorescent derivatives were observed: A, those transported into the cells and subsequently incorporated into neutral lipids and phospholipids, B, fatty acids which were taken up by the cells but not utilized metabolically, and C, fatty acids which were not transported across the cell membrane. Fatty acids of the latter group, except the hydrophobic probe, also contained functional groups such as hydroxy, acetylamino or sulfonylamino. When observed in fluorescence microscopy, cells incubated with group A fatty acids contained intracellular fluorescent granules, whereas those incubated with group B fatty acids showed diffuse fluorescence. HL 60 cells undergo differentiation into granulocytes or macrophages upon treatment with dimethylsulfoxide or a phorbol ester, respectively. When compared to the uninduced cells, the transport of the fluorescent fatty acids or palmitic acid as well as their subsequent incorporation into lipids were considerably lower in the granulocytes and higher in the macrophages. The use of the fluorescent derivatives as a tool for studying transport of fatty acids across the cell membrane is discussed.

Isolation and characterization of HL-60 cell variants with different potentials for spontaneous differentiation.

Although HL-60 cells, an in vitro established cell line derived from a patient with acute promyelocytic leukemia, are blocked at the promyelocytic stage of myeloid differentiation, certain chemicals can induce the cells to undergo terminal differentiation into either granulocytes or macrophages. Moreover, a small fraction of the cell population undergoes differentiation spontaneously without the addition of any inducing agent. In this paper it is demonstrated that this cell line is heterogeneous with respect to the ability of the cells to differentiate spontaneously: some clones (SD+) have a higher tendency to do so than others (SD-). In semi-solid medium, SD+ cells developed diffused colonies containing mature monocytes and macrophages, whereas SD- cells developed compact colonies of promyelocytes. Based on these morphological differences the various clones were isolated and analysed. Although only a small fraction of the population actually became differentiated at any particular time, practically all the cells in the SD+ clones had the potential to differentiate spontaneously. The clones also differ in their response to differentiation inducers; whereas some agents induced complete differentiation in both types of clones, others (e.g. actinomycin C and cytosine arabinoside) induced only SD+ clones, suggesting that differentiation induced by the latter agents is related to the ability of the cells to differentiate spontaneously. Thus the potential of leukemic cells to undergo spontaneous differentiation may be an important factor when considering differentiation-inducing therapy for leukemic patients.

Stimulation of erythroid progenitors by high concentrations of erythropoietin results in normoblasts arrested in G2 phase of the cell cycle.

The cell cycle status of human erythroid precursors generated in a two-step liquid culture was studied by a double-labeling flow cytometric technique. Following a first phase, where peripheral blood mononuclear cells were cultured in the presence of a combination of growth factors, not including erythropoietin (Epo), the cells were washed and recultured in a second phase in the presence of Epo. This procedure resulted in a stimulation of the proliferation and maturation of erythroid precursors. In the presence of optimal concentrations of Epo (2 U/mL), a high percentage (> 40%) of cells were found in the S phase of the cell cycle until day 10. Then, as a result of maturation, the proportion of cells in S gradually decreased, reaching less than 2.0% by day 21. At this time, the culture consisted of > 95% hemoglobin-containing, nonproliferating, orthochromatic normoblasts. Cell cycle analysis of this normoblast population demonstrated a bimodal distribution; while the majority of the cells had a diploid (2C) DNA content, i.e., cells in G1 (or G0) phase, a sizable fraction was tetraploid (4C) corresponding to cells in G2. In contrast, in cultures stimulated with physiological concentrations of Epo (around 50 mU/mL), all the terminally differentiated cells were arrested at the G1 phase. These results suggest that Epo is an essential growth-promoting factor for erythroid precursors, but supraphysiological concentrations, such as present in vivo in severe anemia (e.g., aplastic anemia) or after Epo administration, may be associated with development of normoblasts with abnormal DNA content.

Selective photosensitization of human leukemic cells by a pyrene-containing fatty acid.

Irradiation with long-wave UV light (LUV) at 366 nm of cells that had been incubated with 12-(1-pyrene)dodecanoic acid (P12), a fatty acid derivative with a covalently linked pyrene nucleus, resulted in cytotoxicity. Using the in vitro established human cell lines HL-60 and U-937, we demonstrated that these leukemic cells are much more susceptible to the photosensitizing effect of P12 than normal bone marrow (BM); a 4-log reduction in the number of clonogenic leukemic cells was achieved under conditions where colony formation by normal hemopoietic progenitors was reduced by less than 40%. Moreover, the results of irradiating mixed populations of leukemic and normal cells indicated that phototoxicity of leukemic cells was not affected by the presence of a large excess of normal BM cells, nor was the survival of normal BM cells influenced by the presence of leukemic cells. These findings suggested that the procedure could be adapted for selective ex vivo elimination of malignant cells, i.e., purging of BM in remission prior to autologous transplantation.

Marker analysis of cloned populations of human monocytes.

The presence of myelomonocytic progenitor cells in human peripheral blood was used for the analysis of cloned populations of human monocytes. Colonies of granulocytes and macrophages were obtained by plating peripheral blood mononuclear cells (PBM) in methylcellulose containing medium in the presence of medium conditioned by nonstimulated PBM (CM). Following 20-25 days of incubation, most colonies were found to consist of cells with monocyte-macrophage morphology. Cloned populations of monocytes were tested for several monocyte membrane markers and compared to noncloned adherent monocytes. HLA-DR, 63D3, LeuM2 antigens and Fc receptors were expressed on cells from individual colonies in similar proportions to their expression on noncloned monocytes. Some colonies were uniform in their negative expression of the 63D3 antigen, as were the noncloned monocytes. Although the clonality of cells tested was not directly proven, these results indicated that at least for some monocyte markers, heterogeneous expression was obtained in monoclonal populations of monocytes. It is possible, however, that testing of additional markers and functions may reveal homogeneous clones of monocytes and suggest the existence of stable subsets.