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.

Demonstration of a second pharmacologically active promoter region in the NGF gene that induces transcription at exon 3.

Nerve growth factor (NGF) has been demonstrated to facilitate neurite outgrowth, rescue neurons from injury, and prevent programmed cell death in neurons. However, the therapeutic potential of NGF is limited by metabolic instability and poor CNS penetration. These limitations might be circumvented by identifying compounds which increase endogenous production of NGF in the brain. We sought to determine the site of all pharmacologically inducible promoters in the NGF gene using a differential analysis based on semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). Mouse L929 cells were serum deprived and NGF mRNA was induced by treatment with phorbol 12-myristate 13-acetate (PMA), 1,25-dihydroxy-vitamin D3 (calcitriol) or horse serum. An increase in transcripts initiating at exon 1 was noted in cDNA from cells induced with all three agents. In addition, we also observed an increase in cDNA transcripts that initiate at exon 3 and do not include exons 1 and 2 (4.38 +/- 0.42, 2.56 +/- 0.05 and 3.04 +/- 0.03 fold increase over control for PMA, calcitriol and serum, respectively). Each of these increases was completely inhibited in the presence of actinomycin D, indicating that the increased levels of mRNA were due to increases in transcription and not mRNA stabilization. These results confirm the previous demonstration of a promoter for NGF near exon 1 and establish a pharmacologically inducible promoter in the NGF gene near exon 3 that could be targeted for therapeutic intervention.

[Antimitotic compounds as modifiers of the radiation lesion of cultured cells]. / Antimitoticheskie soedineniia kak modifikatory luchevogo porazheniia kul'tiviruemykh kletok.

The features of antimitotic substances as radioprotectors were studied. In vitro experiments have demonstrated that taxol revealed radioprotective features concerning the process of polymerization of irradiated microtubules. These results were the basis for the use of taxol and some other substances with high affinity for cytoskeleton proteins as potential radiomodificators in vivo. Experiments with cultivated fibroblasts revealed that colchicine significantly enhances radioactive injuries of cells while taxol and phalloidin manifest their radioprotective features.

Induction of cytotoxic factor in mice by lignified materials combined with OK-432 (Picibanil).

Intravenous administration of pine cone lignin-related substance (Fr. VI) significantly stimulated OK-432-elicited cytotoxic factor (CF) production in ICR mouse serum. The level of CF elicited after OK-432 administration peaked after 2 h and declined to basal level within 6 h. The CF productibility depended greatly on both dose and the interval between the administration of the Fr. VI and OK-432. Most natural and synthetic lignins, their degradation products, and polysaccharides, including pine cone hemicellulose fractions, had much weaker CF-inducing (priming) activity. When Fr. VI was treated with NaCl02 to decompose the lignin portion, the priming activity was significantly reduced. The data suggest that the potent priming activity of Fr. VI might de a result of some conjugation between the lignin portion and other components including polysaccharides.

[Cytotoxicity of Corylifoliae fructus. I. Isolation of the effective compound and the cytotoxicity].

The ethanol extract of Psoraleae Fructus (Psoralea corylifolia L.) was found to have cytotoxic activity against L929-cells in cell culture. The active compound was isolated by column chromatography on silica gel and identified as bakuchiol by means of spectral evidence. The cytotoxic activity of bakuchiol in cell culture was observed in short time and found to be unreversible. The mechanism of the cytotoxic activity was considered to be due to an injury of cell membrane from electron microscopic observation and hemolytic activity.

[Cytotoxicity of corylifoliae fructus. II. Cytotoxicity of bakuchiol and the analogues].

Bakuchiol is a major component of Corylifoliae Fructus (Psoralea corylifolia L.) and has been clarified to have cytotoxic activity. The chemical structure-cytotoxic activity relationship of bakuchiol was investigated by means of cytotoxic activity of synthesized analogues of bakuchiol and phenol. It was proved that an alkyl group was necessary for cytotoxic activity. But the double bonds in the unsaturated hydro-carbon group exerted but little influence on the cytotoxic activity. The cytotoxic activity of bakuchiol was the strongest as compared with that of the analogues examined.

[Cytotolerance of bioceramics in cell culture studies]. / Cytotolerance biokeramiky sledovaná v bunecných kulturách.

Biological effects of ceramic implant material of Czechoslovak make prepared on the basis of alumina were studied here. The material was proved to be quite inert both toward epithelioid Hep-2 cells and toward L-line fibroblastoid cells in experiments with nonspecific application, i.e., in cell cultures.

L-929 cells under hyperosmotic conditions. Water, Na+, and K+.

Changes in cell water content resulting from sorbitol addition to the environment of L-929 cells were evaluated gravimetrically using 14C-labeled polyethylene glycol as a probe of extracellular space. Reductions in cell water were proportional to sorbitol supplements up to 0.6 molal, above which no further measurable decrease occurred. No volume regulation occurred for at least 1 h but the percentage of cell water lost was quickly regained when physiological conditions were restored. The amount of cell water lost because of a given hyperosmotic exposure was found to exceed the loss of cell volume. That discrepancy could be the result of an overestimation of extracellular space and/or an underestimation of cell volume reduction as a result of in-folding of the cell surface. Na+ and K+ were also measured in cells of variable water content and volume: no significant change occurred in the amounts of these ions per cell, but large increases in total cell concentration resulted from hyperosmotic exposure. The sum of Na+ and K+ concentrations exceeds the total osmotic pressure of the medium indicating that an appreciable fraction of Na+ and K+ must be bound to fixed charges within the cells. The results are evaluated in the context of intracellular organization.

Glycolysis in permeabilized L-929 cells.

Mouse L-929 cells permeabilized with dextran sulphate (DSP cells) carry out glycolysis when supplemented with glucose, ATP and NAD+ in a suitable incubation buffer. Glycolytic rates were linear and generally independent of cell density over the range examined (1 x 10(6)-10 x 10(6) cells/ml). Electron microscopy revealed characteristic changes in DSP cell ultrastructure, notably for nuclei and mitochondria. Some cells lacked plasma membranes, while others appeared intact. In the latter case, estimates of the lesion size in plasma membranes were obtained from volume of distribution studies using 14C-labelled proteins, and infiltration of fluorescein isothiocyanate dextran. The results indicated the presence of lesions large enough to allow globular proteins of about 400 kDa to cross the cell surface. In spite of that, only about 10% of total cell protein exited from DSP cells during a 30 min incubation period. We propose that none of the glycolytic enzymes in DSP cells can exist completely in solution in the 'cytosol', suggesting extensive enzyme organization. The results are interpreted within the broader picture of metabolic organization in animal cells and the nature of the 'cytosol'.

Comparison of cell death and adenosine triphosphate content as indicators of acute toxicity in vitro.

1. Anchorage-independent LS cells, derived from L929 mouse fibroblasts, were used as an in vitro alternative to animals for the assessment of acute toxicity. The two end points were cell death, indicated by fluorescein diacetate and ethidium bromide, and intracellular adenosine triphosphate (ATP) content. 2. Concentrations of 20 test compounds which produced a 50% decrease in the ATP contents of control cells (ATP50) ranged from 17 micrograms ml-1 for diethylstilboestrol to 7.0 mg ml-1 for sodium chloride. 3. The concentrations which caused 50% cell death ranged from 16 micrograms ml-1 for diethylstilboestrol to 8.0 mg ml-1 for paracetamol. 4. There was a good numerical correlation (r = 0.99) between the ranks of ATP50 and CD50 end-points, there being only minor changes in order between the ranks. 5. The slopes of the dose-response plots for individual chemicals were markedly different.

Cholesterol enhances mouse hepatitis virus-mediated cell fusion.

Mouse hepatitis virus (MHV) infection of the L-2 subline of mouse fibroblasts results in acute infection characterized by extensive cell fusion. In contrast, infection of the LM-K subline leads to virus persistence with reduced cell fusion. We undertook studies designed to elucidate the role of host cell membrane lipid composition and the cytoskeleton in modulating the fusion process and the resultant effect(s) on virus persistence. MHV-induced cell fusion proceeded normally in cells treated with cytoskeleton-disrupting drugs, cytochalasin B and colchicine. Modification of cell membrane fatty acid composition by supplementation of LM-K cells with arachidonic (C-20:4) or palmitic (C-16:0) acids had little effect on the extent of MHV-induced cell fusion or on virus replication. However, supplementation of both cell types with cholesterol (resulting in increased membrane cholesterol/fatty acid ratio) resulted in marked enhancement of virus-mediated cell fusion. The increase in cell membrane cholesterol did not enhance internalization of MHV suggesting that cholesterol primarily modulates a later event. This suggestion was confirmed by demonstrating cholesterol-enhancement of fusion in a contact fusion assay. Cholesterol-supplemented L-2 cells were less productive for virus replication than unsupplemented cells, in agreement with our previous observations that MHV replication is compromised by extensive cytopathic effect. Although cholesterol-supplemented LM-K cells showed increased susceptibility to MHV-mediated cell fusion, the extent of such susceptibility did not approach that observed in L-2 cells. Also, the property of LM-K cells to support MHV persistence was not abolished by cholesterol supplementation. Thus membrane fusion resistance and MHV persistence are modulated but not alleviated by cell membrane cholesterol content.

[Interferon-inducing and antiviral activity of maleic anhydride copolymers]. / Interferonindutsiruiushchaia i protivovirusnaia aktivnost' sopolimerov maleinovogo angidrida.

Data on synthesis of new polymer compounds with potential interferon-inducing and antiviral activity are presented. Maleic anhydride (MA)-based alternating copolymers, in particular, copolymers of divinyl ether with MA containing five-member oxygen cycles in the chain, as well as furan copolymers with MA, were shown to be promising interferon inducers. The antiviral activity was found in copolymers based on allene hydrocarbons.

[Immunomodulating and antitumor activity of polysaccharides of plant origin]. / Immunomoduliruiushchaia i protivoopukholevaia aktivnost' polisakharidov rastitel'nogo proiskhozhdeniia.

The effect of the polysaccharides tagetan, palustran, and zymosan injections to BALB/c mice on the formation of cytotoxic T-lymphocytes in allogenic mixed culture has been studied. Lymphocytes from a 5-day-old mixed culture were characterized by the greatest cytotoxic activity, with spleenocytes serving as reacting cells in mice injected 12-50 mg/kg of tagetan, 20 mg/kg of zymosan and 10-100 mg/kg of palustran, 7, 3 and 5 days, respectively, before the beginning of allogeneic stimulation in vitro. To determine correlation between immunostimulating and antitumor activity 25 mg/kg of tagetan every 14 days, 20 mg/kg of zymosan every 6 days, and 10 mg/kg of palustran every 6 days, were injected to BALB/c and BALB/c nu/nu mice prior to and following subcutaneous implantation of Crocker sarcoma cells. By the end of the first month of immunotherapy the tumour size in mice on tagetan or zymosan was 2-2,5 times smaller than in control animals. Tumour growth inhibition with palustran was about 30%. Polysaccharide administration to BALB/c nu/nu mice was not accompanied by tumour growth inhibition. The data obtained suggest that inhibition of Crocker sarcoma growth in mice injected the above polysaccharides is mediated by stimulation of antitumour T-cell activity.

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.

[Interferon-inducing and antiviral activity of dsRNA of yeast origin]. / Interferonindutsiruiushchaia i protivovirusnaia aktivnost' dsRNK drozhzhevogo proiskhozhdeniia.

The paper describes derivation of double-stranded RNA from K plasmid of Sachcaromyces cerevisiae yeast by means of electrophoresis in agar gel and by differential salting out with 4 M lithium chloride. Studies in vitro and in vivo demonstrated a high interferon-inducing and antiviral activity of dsRNA preparations from the yeast plasmid.

Saturable attachment sites for type 3 mammalian reovirus on murine L cells and human HeLa cells.

Attachment of [35S]methionine-labelled mammalian type 3 reovirus to murine L cells and human HeLa cells was studied under equilibrium conditions. Cellular attachment sites could be completely saturated with 35S-labelled reovirus, indicating that specific attachment sites for reovirus are present on the surface of these cells. We calculated that L cells possess about 86000-105000 attachment sites per cell while HeLa cells possess about 126000-147000 sites per cell for type 3 reovirus. Unlabelled reovirus was highly efficient in competing for attachment by 35S-labelled reovirus to the saturable attachment sites of both L and HeLa cells, further indicating the specificity of the interaction. We also found that unlabelled reovirus competed equally well for both binding and internalization of 35S-labelled reovirus into murine L cells, suggesting that the L cell attachment site may serve as a virus entry site. Phospholipase digestion of L cells had no effect on subsequent reovirus attachment, while treatment of L cells with moderate concentrations of bromelain (but not trypsin, proteinase K or pronase) and Vibrio cholerae neuraminidase reproducibly decreased subsequent reovirus attachment. These results and those of others (Epstein et al., 1984, Virology 133, 46-55) suggest that mammalian reoviruses attach to specific cell surface receptors on at least two species of mammalian cells to initiate the infectious cycle.

Antiviral activities of cloned human leukocyte interferons against herpes simplex virus type 2 infections of mice.

Human alpha-interferons (IFN-alpha s) made in bacteria were examined for antiviral activity against herpes simplex virus type 2 (HSV-2) infections of mouse L-cells in vitro, and acute cervicovaginal and lethal systemic HSV-2 infections of BALB/c mice. The recombinant DNA-derived hybrid interferon IFN-alpha AD(Bgl) showed pronounced antiviral activity in vitro, exceeding the activity of either of the parental subtypes IFN-alpha A and IFN-alpha D and that of the other hybrids IFN-alpha AD(Pvu) and IFN-alpha DA(Bgl). A combination of topical and systemic treatments with IFN-alpha A and IFN-alpha AD(Bgl) failed to protect mice from subsequent challenge with an acute cervicovaginal infection of HSV-2. Protection from lethal systemic HSV-2 infection in mice was observed when IFN-alpha AD(Bgl) and IFN-alpha AD(Pvu) were administered systemically, whereas IFN-alpha A failed to confer protection. These results suggest that for protection against infection with HSV-2, the routes of introduction of the virus and of the interferon influence the host response to interferon therapy.

New selenium-containing silver amalgam.

An investigation was made to determine the effects of the addition of selenium to the silver-tin amalgam alloy. The addition of 0.2 wt% to 0.6 wt% selenium in the alloy completely eliminated the cytotoxicity, evaluated by the 51Cr release assay and morphology, of the silver amalgam on the L cells and JTC-12 cells. The physical properties of the amalgam containing 0.2 wt% selenium in the alloy were tested according to the American Dental Association Specification No. 1 for Alloy for Dental Amalgam and were found to meet the requirements.