Phototoxic effect of curcumin on methicillin-resistant Staphylococcus aureus and L929 fibroblasts.

Photodynamic therapy has been investigated as an alternative method of killing pathogens in response to the multiantibiotic resistance problem. This study evaluated the photodynamic effect of curcumin on methicillin-resistant Staphylococcus aureus (MRSA) compared to susceptible S. aureus (MSSA) and L929 fibroblasts. Suspensions of MSSA and MRSA were treated with different concentrations of curcumin and exposed to light-emitting diode (LED). Serial dilutions were obtained from each sample, and colony counts were quantified. For fibroblasts, the cell viability subsequent to the curcumin-mediated photodynamic therapy was evaluated using the MTT assay and morphological changes were assessed by SEM analysis. Curcumin concentrations ranging from 5.0 to 20.0 µM in combination with any tested LED fluences resulted in photokilling of MSSA. However, only the 20.0 µM concentration in combination with highest fluence resulted in photokilling of MRSA. This combination also promoted an 80% reduction in fibroblast cell metabolism and morphological changes were present, indicating that cell membrane was the main target of this phototherapy. The combination of curcumin with LED light caused photokilling of both S. aureus strains and may represent an alternative treatment for eradicating MRSA, responsible for significantly higher morbidity and mortality and increased healthcare costs in institutions and hospitals.

L-Cell Expression of Melanocortin-4-Receptor Is Marginal in Most of the Small Intestine in Mice and Humans and Direct Stimulation of Small Intestinal Melanocortin-4-Receptors in Mice and Rats Does Not Affect GLP-1 Secretion.

The molecular sensors underlying nutrient-stimulated GLP-1 secretion are currently being investigated. Peripheral administration of melanocortin-4 receptor (MC4R) agonists have been reported to increase GLP-1 plasma concentrations in mice and humans but it is unknown whether this effect results from a direct effect on the GLP-1 secreting L-cells in the intestine, from other effects in the intestine or from extra-intestinal effects. We investigated L-cell expression of MC4R in mouse and human L-cells by reanalyzing publicly available RNA sequencing databases (mouse and human) and by RT-qPCR (mouse), and assessed whether administration of MC4R agonists to a physiologically relevant gut model, isolated perfused mouse and rat small intestine, would stimulate GLP-1 secretion or potentiate glucose-stimulated secretion. L-cell MC4R expression was low in mouse duodenum and hardly detectable in the ileum and MC4R expression was hardly detectable in human L-cells. In isolated perfused mouse and rat intestine, neither intra-luminal nor intra-arterial administration of NDP-alpha-MSH, a potent MC4R agonist, had any effect on GLP-1 secretion (P ≥0.98, n = 5-6) from the upper or lower-half of the small intestine in mice or in the lower half in rats. Furthermore, HS014-an often used MC4R antagonist, which we found to be a partial agonist-did not affect the glucose-induced GLP-1 response in the rat, P = 0.62, n = 6). Studies on transfected COS7-cells confirmed bioactivity of the used compounds and that concentrations employed were well within in the effective range. Our combined data therefore suggest that MC4R-activated GLP-1 secretion in rodents either exclusively occurs in the colon or involves extra-intestinal signaling.

A human inhibitor of tumor necrosis factor alpha.

Urine of some febrile patients exhibits a TNF-alpha inhibitory activity (TNF-alpha INH), sensitive to heat and trypsin, with an apparent mol wt of 40-60 X 10(3) and a pI range of 5.5-6.1. As for the Il-1 INH, the TNF INH activity involves a competitive mechanism of action suggesting the existence of a family of negative feedback-regulating molecules interfering with cytokines actions.

Nonhematopoietic cells selected for resistance to tumor necrosis factor produce tumor necrosis factor.

TNF-resistant lines of L cells can be derived from TNF-sensitive populations by repeated exposure to TNF, and these resistant L cells, in contrast to sensitive L cells and other types of cells, lack demonstrable cell surface receptors for TNF. We have now found that TNF-resistant L cells produce a factor that is cytotoxic for L cells and has the following distinguishing characteristics of mouse TNF: it is a protein of 43 kD, composed of 16 kD subunits, that competes with TNF for receptor binding, induces hemorrhagic necrosis of the TNF-sensitive mouse sarcoma Meth A, has synergistic cytotoxic action with interferon, and its activity is neutralized by antibody to TNF. The two conclusions of this study are that cells selected for TNF resistance spontaneously produce a molecule resembling macrophage TNF, and that cells of nonhematopoietic origin are capable of producing TNF.

Effects of ethidium bromide on the cytochrome content and ultrastructure of L cell mitochondria.

Ethidium bromide intercalates between the bases of native DNA, resulting in several biological anomalies. The effects of ethidium bromide on the mitochondria of cultured mouse L cells were studied. At a concentration of 1 microg ethidium bromide/ml it was observed that concentrations of cytochromes a + a(3) and b decreased, a + a(3) more rapidly than b. In contrast, the concentration of cytochromes c(1) and c increased or remained the same as in control cells. Concomitant with the decrease of cytochromes a + a(3) and b was an enlargement of the mitochondria and a reduction in the cristae. The cristae that remained were abnormally organized. After prolonged treatment with ethidium bromide a second population of small, more normally organized mitochondria was apparent. These effects of ethidium bromide could be reversed.

Effect of acid pH on macromolecular synthesis in L cells.

Eagle's medium adjusted to pH 6 was found to inhibit the rate of RNA and protein syntheses in monolayer cultures of L cells. Incubation of the cells at pH 6 decreased the rate of incorporation of amino acids into nascent peptide chains and caused a disaggregation of polyribosomes. Messenger RNA seemed to persist during the exposure of the cells to medium adjusted to pH 6, since protein synthesis resumed when the cells were transferred to recovery medium containing actinomycin D. The inhibitory effects of pH 6 on macromolecular synthesis were reversible and the viability of the cells exposed to pH 6 did not decrease. The permeability of the cells was not altered by the exposure to pH 6.

Independence of centriole formation and DNA synthesis.

The temporal relationship between cell cycle events and centriole duplication was investigated electron microscopically in L cells synchronized by mechanically selecting mitotic cells. The two mature centrioles which each cell received at telophase migrated together from the side of the telophase nucleus distal to the stem body around to a region of the cytoplasm near the stem body and then into a groovelike indention in the early G(1) nucleus, where they were found throughout interphase. Procentrioles appeared in association with each mature centriole at times varying from 4 to 12 h after mitosis. Since S phase was found to begin on the average about 9 h after mitotic selection, it appeared that cells generated procentrioles late in G(1) or early in S. During prophase, the two centriolar duplexes migrated to opposite sides of the nucleus and the daughter centrioles elongated to the mature length. To ascertain whether any aspect of centriolar duplication was contingent upon nuclear DNA synthesis, arabinosyl cytosine was added to mitotic cells at a concentration which inhibited cellular DNA synthesis by more than 99%. Though cells were thus prevented from entering S phase, the course of procentriole formation was not detectibly affected. However, cells were inhibited from proceeding to the next mitosis, and the centriolar elongation and migration normally associated with prophase did not occur.

Biochemical studies on cell fusion. II. Control of fusion response by lipid alteration.

The preceding communication (Roos, D.S. and P.W. Choppin, 1985, J. Cell Biol. 101:1578-1590) described the lipid composition of a series of mouse fibroblast cell lines which vary in susceptibility to the fusogenic effects of polyethylene glycol (PEG). Two alterations in lipid content were found to be directly correlated with resistance to PEG-induced cell fusion: increases in fatty acyl chain saturation, and the elevation of neutral glycerides, including an unusual ether-linked compound. In this study, we have probed the association between lipid composition and cell fusion through the use of fatty acid supplements to the cellular growth medium, and show that the fusibility of cells can be controlled by altering their acyl chain composition. The parental Clone 1D cells contain moderately unsaturated fatty acids with a ratio of saturates to polyunsaturates (S/P) approximately 1 and fuse virtually to completion following a standard PEG treatment. By contrast, the lipids of a highly fusion-resistant mutant cell line, F40, are highly saturated (S/P approximately 4). When the S/P ratio of Clone 1D cells was increased to approximate that normally found in F40 cells by growth in the presence of high concentrations of saturated fatty acids, they became highly resistant to PEG. Reduction of the S/P ratio of F40 cells by growth in cis-polyunsaturated fatty acids rendered them susceptible to fusion. Cell lines F8, F16, etc., which are normally intermediate between Clone 1D and F40 in both lipid composition and fusion response, can be altered in either direction (towards either increased or decreased susceptibility to fusion) by the addition of appropriate fatty acids to the growth medium. Although trans-unsaturated fatty acids have phase-transition temperatures roughly similar to saturated compounds, and might therefore be expected to affect membrane fluidity in a similar manner, trans-unsaturated fatty acids exerted the same effect as cis-unsaturates on the control of PEG-induced cell fusion. This observation suggests that the control of cell fusion by alteration of fatty acid content is not due to changes in membrane fluidity, and thus that the fatty acids are involved in some other way in the modulation of cell fusion.

Selection of mammalian cells resistant to a chloramphenicol analog.

This study describes the selection and preliminary characterization of mammalian cells resistant to 100 mug Tevenel/ml. Tevenel, the sulfamoyl analog of chloramphenicol, is a specific inhibitor of mitochondrial protein synthesis. After growth in suspension culture for 5 days in 100 mug Tevenel/ml and subsequent plating in 100 mug Tevenel/ml, LMTK- cells yielded resistant clones. As a control, L cells treated identically yielded no clones. Three resistant clones were chosen for study. Each resistant cell line had an identical growth rate in the presence and absence of 100 mug Tevenel/ml. By plating efficiency analysis, the resistant cells were found to be cross-resistant to D-chloramphenicol. The change responsible for resistance was found to be stable for at least 100 generations in the absence of the drug. Protein synthesis by isolated mitochondria of resistant cells was found to be less inhibited by concentrations of both Tevenel and D-chloramphenicol up to 200 mug/ml than the protein synthesis by LMTK- mitochondria. This resistance in vitro was not changed by incubation of the mitochondria in 0.01% Triton X-100.

Biochemical studies on cell fusion. I. Lipid composition of fusion-resistant cells.

A series of stable cell mutants of mouse fibroblasts were previously isolated (Roos, D. S. and R. L. Davidson, 1980, Somatic Cell Genet., 6:381-390) that exhibit varying degrees of resistance to the fusion-inducing effect of polyethylene glycol (PEG), but are morphologically similar to the parental cells from which they were derived. Biochemical analysis of these mutant cell lines has revealed differences in whole cell lipid composition which are directly correlated with their susceptibility to fusion. Fusion-resistant cells contain elevated levels of neutral lipids, particularly triglycerides and an unusual ether-linked lipid, O-alkyl, diacylglycerol. This ether lipid is increased approximately 35-fold over parental cells in the most highly PEG-resistant cell line. Fusion-resistant cells also contain more highly saturated fatty acyl chains (ratio of saturated to polyunsaturated fatty acids [S/P ratio] approximately 4:1) than the parental line (S/P ratio approximately 1:1). Cells which are intermediate in their resistance to PEG have ether lipid and fatty acid composition which is intermediate between the parental cells and the most fusion-resistant mutants. In a related communication (Roos, D. S. and P. W. Choppin, 1985, J. Cell. Biol., 100:1591-1598) evidence is presented that alteration of lipid content can predictably control the fusion response of these cells.

Serum factors alter the extent of dephosphorylation of ligands endocytosed via the mannose 6-phosphate/insulin-like growth factor II receptor.

Mouse L-cells that contain the cation-independent (CI) mannose 6-phosphate (Man 6-P)/insulin-like growth factor (IGF) II receptor endocytose acid hydrolases and deliver these enzymes to lysosomes. The postendocytic loss of the Man 6-P recognition marker from the cell-associated acid hydrolases was assessed by CI-Man 6-P receptor affinity chromatography. 125I-labeled acid hydrolases internalized by L-cells grown at high density were delivered to lysosomes but were not dephosphorylated. In contrast, the same 125I-labeled hydrolases internalized by L-cells maintained at low density were delivered to lysosomes and were extensively dephosphorylated. The dephosphorylation at low density required 5 h for completion suggesting that the phosphatase responsible for the dephosphorylation is located within the lysosomal compartment. Transition from the high to low density state was rapid and was not inhibited by cycloheximide. Medium substitution experiments indicated that serum factors were necessary to maintain the L-cells in the dephosphorylation-competent (low density) state, and that serum-free conditions led to a dephosphorylation-incompetent (high density) state. Addition of IGF II to cells in serum-free medium allowed acid hydrolases subsequently introduced by endocytosis to be dephosphorylated. The results indicate that the removal of the Man 6-P recognition marker from endocytosed acid hydrolases is regulated by serum factors in the growth medium, including IGF II.

Enhancement of Sendai virus-mediated cell fusion by cupric ions.

The effect of divalent cations on cell fusion by concentrated Sendai virus, inactivated by beta-propiolactone, was investigated using Vero and mouse L-929 cells in monolayers. With both cell lines, which are normally resistant to exogenous viral fusion, Cu(2+) in sublethal concentrations was found to promote polykaryon formation to a marked degree. The simultaneous presence of Cu(2+) and virus was required for this effect, which was thought to be related to the cytotoxic action of Cu(2+) on the cell membrane. Accordingly, under standard conditions and in the absence of virus, leakage of isotopically labeled intracellular protein was shown to bear a quantitative relationship to Cu(2+) concentration. Concomitant changes in the membrane were seen electron microscopically to consist of loss of microvilli and the appearance of numerous vesicles on, or adjacent to, the membrane. The relationship of enhanced fusibility to these toxic changes was not further elucidated. The fusion-promoting effect of Cu(2+) far exceeded that of Ca(2+); and other cations tested had no effect.

On the recovery of transcription after inhibition by actinomycin D.

After pulse exposure to concentrations of actinomycin D (AMD) sufficient to abolish transcription, Vero cells recover RNA synthesis much more rapidly than most other cell types. This is only in part attributable to the remarkable capacity of Vero very promptly to excrete bound AMD, elimination of which, although necessary, is not a sufficient condition for resurgence of RNA synthesis. After elimination of higher concentrations of AMD from Vero, although over-all RNA synthesis resumes a normal rate within 24 hr, protein synthesis lags, and a long period of division-delay ensues. Division-delay lasting 2-3 days results from exposure of Vero to doses of AMD greater than those that suppress RNA synthesis by greater than 90% (e.g. 1 microg/ml for 2 hr) but not by lower doses, which permit almost immediate reentry into the cell cycle. In contrast, although L cells recover over-all RNA synthesis very slowly after pulse treatment with AMD, resumption of protein synthesis or cell division is not comparably delayed thereafter. These and other data suggest that the early restoration of RNA synthesis in Vero after relief of inhibition by AMD is qualitatively imperfect. The results reported herein are explainable by the hypothesis that the synthesis of those species of RNA which are involved, directly or indirectly, in reactivating the transcription of genes controlling progression in the cell cycle is relatively resistant to suppression by AMD. Decay of such RNA templates and their products, which differs in different cell types during inhibition by AMD, determines the duration of division-delay.

Respiratory enzymes and mitochondrial morphology of HeLa and L cells treated with chloramphenicol and ethidium bromide.

Exposure of HeLa and L cells to chloramphenicol causes a progressive dose-dependent decrease in cytochrome oxidase and succinate-cytochrome c reductase activities, concomitant with an increase in the amount of cytochrome c. At 2-3 days, the specific activities of the enzymes have fallen to about one-half of control values; the mitochondria appear swollen. By day 5, enzyme activities are about one-quarter of control values; the mitochondria are more swollen, with disorientation and disintegration of cristae. By day 6-8, after three generations, growth has stopped, enzyme activities are approximately the same as on day 5, and cytochrome c content has reached 170% of control value. Mitochondria show severe changes, cristae being affected more than peripheral inner membrane. The number of profiles continues to be nearly normal. After 30 days, cytochrome oxidase activity remains low but now there are mitochondria in intermediate and condensed configuration. There is a gradual accumulation in the cytoplasm of smooth membrane elements. If chloramphenicol is removed, cells recover. Ethidium bromide treatment for up to 8 days yields results virtually identical to those obtained with chloramphenicol. Cells treated with 10(-4)M KCN show a decrease in cytochrome oxidase activity to about one-third of control value and an elevated amount of cytochrome c. Only a small number of mitochondria appear damaged. Autochthonous mitochondrial syntheses appear to be essential for the organization of the cristae. When cytochrome oxidase activity is impaired, a regulatory mechanism for cytochrome biosynthesis geared to mitochondrial function may be lacking, resulting in an increase in cytochrome c content.

The fine structure and immunological labeling of the achromatic mitotic apparatus after disruption of cell membranes.

After treatment of HeLa and L cells with vinblastine sulfate the material of microtubules (tubulin) was reorganized into (a) large paracrystals (PC) of tightly packed tubules; (b) smaller aggregates of tubules with greater diameter whose walls are constituted from well defined, helically arranged morphological subunits; and (c) microtubules associated with helices of polyribosomes of uniform size. All of these structures survived disruption of cellular membranes by means of a nonionic detergent. Following a thorough stripping of membranes there remained a subcellular fraction sedimenting at 1,500 g for 15 min, in which were contained nuclei, centrioles, and the above mentioned microtubular elements, maintained as a complex of organelles by an interconnecting network of 80 A microfibrils. As a result of membrane disruption it was possible to localize precisely in the electron microscope the binding of ferritin antibody conjugates. Specific labeling at the surface of PC and microtubule aggregates could be demonstrated. This result was substantiated by means of the immunoperoxidase method of labeling the PC. A concentrated deposit of ferritin was also found in the vicinity of centrioles and related structures, the annuli of the nuclear pore complex and the annulate lamellae. However, the specificity of the label on these organelles remains questionable because ferritin, albeit in lower concentration, was also present on them in control preparations reacted with preimmune sera.

Modulation of NCAM expression by transforming growth factor-beta, serum, and autocrine factors.

The expression of NCAM (neural cell adhesion molecule) is precisely regulated in terms of cell type specificity and developmental control. We searched for extracellular factors that may be involved in this regulation using N2A neuroblastoma and NIH 3T3 fibroblastic cells. Factors contained in FBS promoted a two- to threefold increase in NCAM protein and mRNA abundance in both cell lines. This increase in NCAM expression in high serum could be entirely attributed to enhanced levels of the NCAM-140 message. Modulation of NCAM synthesis via an autocrine mechanism is suggested by the observation that medium conditioned by N2A cells stimulated NCAM mRNA expression by 3T3 and N2A cells. Among the pure factors tested, transforming growth factor-beta (TGF beta) was found to act as an inducer of NCAM expression in 3T3 but not in N2A cells. 3T3 cells responded to exposure to TGF beta with a two- to threefold increase in NCAM protein and mRNA. Exposure of early-passage embryonic cells to TGF beta resulted in four- and twofold increases in NCAM protein and mRNA abundance, respectively, suggesting a role for TGF beta in modulating NCAM expression in the embryo. TGF beta seems to act by stimulating the transcriptional activity of the NCAM gene because it did not affect transcript stability and stimulated transcription from a proximal promoter element of the NCAM gene.

Cell-cell contacts mediated by E-cadherin (uvomorulin) restrict invasive behavior of L-cells.

L-cells were cotransfected with plasmids coding for mouse E-cadherin (uvomorulin) and the neophosphotransferase gene, and stable transfectants expressing E-cadherin at the cell surface were selected and cloned. Control transfection was done with the neophosphotransferase gene alone. The invasive migration of transfected and untransfected L-cells into three-dimensional collagen gels was then analyzed. L-cells not expressing E-cadherin migrated efficiently into the gels, whereas invasion of the E-cadherin-expressing L-cells was restricted in a cell density dependent manner. At sparse density, when the cells exhibited little cell-cell contacts, no difference was observed between the level of invasion of the cadherin-expressing cells and the control cells. However, with increasing cell density, decreasing amounts of the cadherin-expressing cells but increasing amounts of the control cells migrated into the gels. At confluent density hardly any cadherin-expressing cells were able to migrate into the gels. The inhibition of the invasion of the cadherin-expressing cells could be reverted if confluent cells were cultured in the presence of monoclonal antibodies against E-cadherin. Since the expression of E-cadherin did not influence the invasive mobility of single cells, these results indicate that E-cadherin-mediated cell-cell contacts inhibited invasive cellular migration. Time-lapse videoscopy and studies of cell migration from a monolayer into a cell-free area demonstrated that the restricted invasion could be explained by contact inhibition of cell movement of the cadherin-expressing cells.

Acetylcholine responses in L cells.

L cells, a family of continuous cell lines of mouse fibroblastic origin, generate a prolonged active membrane hyperpolarization (the hyperpolarizing activation response) when stimulated mechanically or electrically. lontophoretically applied acetylcholine elicits a similar response; atropine blocks the acetylcholine but not the electrically or mechanically elicited responses. The hyperpolarizing activation response can also be elicited by electrical, mechanical, or acetylcholine stimulation of cells adjacent to the recorded cell. Propagation of the response from one cell to another is not dependent on direct electrical coupling between cells and is not blocked by application of a bath containing atropine or curare. These results show that L cells are capable of generating an active electrical response. that they are sensitive to at least one neurotransmitter (acetylcholine), and that humorally mediated interaction (probably noncholinergic) between L cells occurs.

Induction of porcine regulatory cells by mycophenolic Acid-treated dendritic cells.

Tolerance induction in murine allogeneic transplantation is relatively easy, often by induction of regulatory T cells (Treg). Unfortunately, the implementation of these models in clinical situations has not yielded reliable protocols of tolerance induction in humans. Our project sought to create a preclinical model of tolerance induction in large animals. Our current efforts seek to induce and characterize porcine Treg, obtaining dendritic cells (DC) able to preferentially stimulate them. DCs were differentiated from blood monocytes with porcine recombinant interleukin-4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) for 6 days. These DCs were then stimulated by human CD40 ligand-transfected L cells with or without mycophenolic acid (MPA) for 48 hours. We analyzed surface marker expression, cytokine synthesis, and ability to stimulate allogeneic peripheral blood mononuclear cells (PBMC). The porcine lymphocytes underwent 4 rounds of 1-week stimulation with allogeneic DC treated or not with MPA. At the end of this coculture we analyzed their capacity to suppress allogeneic PBMC proliferation induced by mature DC. Our results showed that porcine DCs pretreated with MPA display a low expression of B7 costimulatory molecules, produce low levels of IL-12, and induce weak proliferation of allogeneic lymphocytes. Moreover, after 4 rounds of stimulation with MPA-treated DCs, PBMCs were able to inhibit an alloreactive response. These preliminary results suggested induction of a regulatory T-cell population that we are currently seeking to characterize.

Determination of rupatadine in pharmaceutical formulations by a validated stability-indicating MEKC method.

A stability-indicating MEKC was developed and validated for the analysis of rupatadine in tablet dosage forms, using nimesulide as internal standard. The MEKC method was performed on a fused-silica capillary (50 microm id; effective length, 40 cm). The BGE consisted of 15 mM borate buffer and 25 mM anionic detergent SDS solution at pH 10. The capillary temperature was maintained at 35 degrees C and the applied voltage was 25 kV. The injection was performed using the hydrodynamic mode at 50 mbar for 5 s, with detection by photodiode array detector set at 205 nm. The method was linear in the range of 0.5-150 microg/mL (r2=0.9996). The specificity and stability-indicating capability of the method were proven through degradation studies inclusive by MS, and showing also that there was no interference of the excipients and no increase of the cytotoxicity. The accuracy was 99.98% with bias lower than 1.06%. The LOD and LOQ were 0.1 and 0.5 microg/mL, respectively. The proposed method was successfully applied for the quantitative analysis of rupatadine in pharmaceutical formulations, and the results were compared to a validated RP-LC method, showing non-significant difference (p>0.05).