Effect of dietary oils on the production of n-3 and n-6 metabolites of leukocyte 5-lipoxygenase in five rat strains.

We examined the influence of various dietary oils, including linseed and fish oil on the relative rates of leukotriene B4 (LTB4) and LTB5 production by rat peritoneal exudate cells in five rat strains. While there was an association between the membrane phospholipid levels of the fatty acid precursors (arachidonic acid (AA) and eicosapentaenoic acid (EPA)) and the rate of synthesis of their respective 5-lipoxygenase products (LTB4 and LTB5), the rate of LTB4 synthesis was a combined function of both AA and EPA levels. We observed a strong linear relationship (correlation coefficient = 0.99) between the ratio of EPA/AA in the cell membrane phospholipids and the ratio of LTB5/LTB4 produced by these cells in vitro; this association was independent of genetic (strain) variability and was independent of the source of EPA (dietary EPA or EPA endogenously synthesized from dietary alpha-linolenic acid).

Flux of intracellular labile zinc during apoptosis (gene-directed cell death) revealed by a specific chemical probe, Zinquin.

BACKGROUND: The transition metal Zn(II) is thought to regulate cell and tissue growth by enhancing mitosis (cell proliferation) and suppressing the counterbalancing process of apoptosis (gene-directed cell death). To investigate the role of Zn(II) further, we have used a UV-excitable Zn(II)-specific fluorophore, Zinquin. The ester group of Zinquin is hydrolyzed by living cells, ensuring its intracellular retention; this allows the visualization and measurement of free or loosely-bound (labile) intracellular Zn(II) by fluorescence video image analysis or fluorimetric spectroscopy. RESULTS: Here we show that in cells undergoing early events of apoptosis, induced spontaneously or by diverse agents, there is a substantial increase in their Zinquin-detectable Zn(II). This increase occurred in the absence of exogenous Zn(II) and before changes in membrane permeability, consistent with a release of Zn(II) from intracellular stores or metalloproteins rather than enhanced uptake from the medium. We propose that there is a major redistribution of Zn(II) during the induction of apoptosis, which may influence or precipitate some of the later biochemical and morphological changes. CONCLUSIONS: The phenomenon of Zn(II) mobilization, revealed by Zinquin, presents a new element in the process of apoptosis for investigation and may permit rapid and sensitive identification of apoptotic cells, particularly in those tissues where their frequency is low.

Phorbol 12-myristate 13-acetate, A23187 and L-adrenaline inhibit phospholipid methylation in human monocytes and lymphocytes. Inhibition is independent of oxyradical production and phospholipid hydrolysis.

The Ca++ ionophore A23187 and phorbol 12-myristate 13-acetate (PMA) caused dose-dependent inhibition of phospholipid (PL) methylation in unfractionated mononuclear cells (MNC), monocytes, and lymphocytes as measured by incorporation of 3H-methyl-groups from [3H-methyl]-L-methionine into phosphatidylcholine (PC), dimethyl phosphatidylethanolamine (PE), and monomethyl PE. This inhibitory effect did not correlate with monocyte superoxide release and was unaltered by the presence of either catalase and superoxide dismutase or the NADPH oxidase inhibitor, diphenylene iodonium (DPI), indicating that oxyradical-mediated oxidation of methionine was not the major cause of inhibition of PL methylation. Furthermore L-adrenaline, which elevates cAMP and does not stimulate superoxide release, also inhibited PL methylation. Inhibition by PMA was not due to reduction in intracellular levels of methionine or S-adenosyl methionine. A23187 caused reduction of S-adenosyl methionine levels only at 1 microM, and had no effect at lower concentrations. Inhibition of PL methylation was shown not to be due to phospholipase A2-dependent hydrolysis of newly methylated PL. Attempts to reverse the inhibitory effect of either A23187 or PMA with the putative protein kinase inhibitors W-7 and H-7 were inconclusive. The mechanism of inhibition of PL methylation by A23187 and PMA remains unclear, but does not appear to be due to oxidation of methionine or hydrolysis of newly methylated PL.

Synergy between zinc and phorbol ester in translocation of protein kinase C to cytoskeleton.

Protein kinase C was measured in the cytoskeletal fraction of lymphocytes, platelets and HL60 cells, by specific binding of [3H]phorbol dibutyrate and by immunoblotting with antibody to a consensus sequence in the regulatory domain of alpha-, beta- and gamma-isozymes of protein kinase C. Treatment of cells for 40 min with a combination of zinc (2-50 microM), zinc ionophore pyrithione and unlabelled phorbol dibutyrate (200 nM) caused up to a ten-fold increase in cytoskeletal protein kinase C and a corresponding decrease in other cellular compartments. Omission of any of the reagents resulted in much less or no translocation. These effects were inhibited by 1,10-phenanthroline, which chelates zinc, and were not seen with calcium. Increase in cytoskeletal protein kinase C persisted for several hours and appeared to involve attachment of the enzyme to actin microfilaments. We propose that zinc, like calcium, regulates the distribution of PKC in cells. However, unlike calcium which controls the binding of PKC to the lipid component on cell membranes, zinc controls the distribution of PKC to membrane cytoskeleton, possibly actin.

Video image analysis of labile zinc in viable pancreatic islet cells using a specific fluorescent probe for zinc.

We used an intracellular zinc-specific fluorophore, Zinquin, in conjunction with fluorescence video image analysis, to reveal labile zinc in pancreatic islet cells, which concentrate this metal for use in synthesis, storage, and secretion of insulin. Zinquin vividly demonstrated zinc in the islet cell secretory granules, which formed a brightly labeled crescent in the cytoplasm between one side of the nucleus and the plasma membrane. Lower but still appreciable amounts of zinc were detected in the remaining cytoplasm, but there was little labeling in the nucleus. Fluorescence intensity varied among islet cells, suggesting differences in zinc content. Their average fluorescence intensity greatly surpassed that of the surrounding pancreatic acinar cells in frozen sections of pancreas and in all other types of cell studied, including lymphocytes, neutrophils, fibroblasts, and erythrocytes. Less labile zinc was detected in cells of the mouse insulinoma cell line NIT-1, regardless of whether they were maintained in long-term culture in the presence or absence of exogenous extracellular zinc. Exposure of islet or insulinoma cells to a high concentration of glucose or other secretagogue decreased the content of labile zinc. Zinquin should be a useful probe for revealing changes in zinc homeostasis in islet B-cells that may be important in their dysfunction and death during diabetes.

Auranofin stimulates LTA hydrolase and inhibits 5-lipoxygenase/LTA synthase activity of isolated human neutrophils.

The effect of auranofin on the 5-lipoxygenase pathway was studied in human neutrophils stimulated with either fMLP or A23187 (with or without arachidonic acid). The synthesis of leukotriene B4 (LTB4), 5-HETE and the all-trans isomers of LTB4 was measured by HPLC. At low concentrations (0.5-2.0 microM), auranofin stimulated LTB4 synthesis, but inhibited it at higher concentrations (100% inhibition at less than 10 microM). In contrast auranofin caused dose-dependent inhibition of the synthesis of 5-HETE and the all-trans isomers of LTB4. Similar observations were made with each agonist. The stimulation of LTB4 synthesis and inhibition of the trans isomer production suggests that auranofin at low concentrations stimulates LTA hydrolase--the enzyme that converts LTA4 to LTB4, whereas the inhibition of synthesis of all lipoxygenase products at higher auranofin concentrations, suggests inhibition of 5-lipoxygenase/LTA synthase.

Differential effects of mepacrine, chloroquine and hydroxychloroquine on superoxide anion generation, phospholipid methylation and arachidonic acid release by human blood monocytes.

The 4-aminoquinolines chloroquine (CQ) and hydroxychloroquine (HCQ), and previously the 9-aminoacridine mepacrine (quinacrine) (MP), have been widely used in the treatment of inflammatory disorders such as rheumatoid arthritis and systemic lupus erythematosus. Their effects are believed to be mediated through phagocytic cells but the precise biochemical basis remains uncertain. We have investigated the effects of these drugs on monocyte superoxide anion (SO) generation elicited by 5 different stimuli-opsonised zymosan (STZ), FMLP, A23187, TPA and fluoride--and sought correlations with effects on two processes which have been linked with monocyte activation, namely arachidonate (AA) release and transmethylation of phosphatidyl ethanolamine (PE) to phosphatidylcholine (PC). In all experiments conditions were adjusted to achieve leucocyte concentrations of drug comparable to those found during in vivo therapy. Neither CQ nor HCQ had any marked effect on SO release induced by TPA, A23187 or fluoride ion, excluding a significant effect on protein kinase C (PKC), calmodulin-dependent kinase(s) or the membrane-bound, superoxide generating NADP(H) oxidase. In contrast MP inhibited the response to TPA and A23187. Each drug also had different effects on surface receptor-dependent responses; thus HCQ inhibited FMLP- but not STZ-induced SO release, whereas CQ and MP inhibited the response to both stimuli. Each drug also displayed different effects on AA release and phospholipid (PL)-methylation; MP and HCQ, but not CQ, inhibited STZ-stimulated AA release while MP and CQ but not HCQ inhibited basal rates of PL-methylation in mononuclear cells (MNC). However, only MP inhibited PL-methylation in an enriched monocyte population. We conclude that despite their close structural similarity, MP, CQ and HCQ each have different metabolic effects and their actions cannot simply be attributed to inhibition of lysosomal functions. Other possible mechanisms of action are discussed. The selective effects of each drug also provide further evidence for multiple pathways of monocyte activation.

Evaluation of a dielectrophoretic bacterial counting technique.

Dielectrophoresis, an electrokinetic migration of particles, can occur in non-uniform alternating electric fields and is dependent upon the dielectric nature of the cells and their suspending medium. An enumeration system utilising this phenomenon is described, which has the potential to count particles selectively, including different bacterial or eukaryotic cell species and even sub-populations of different cell viability states or sizes. Relationships were observed between suspension concentrations and the extent of dielectrophoretic (DEP) collection for polystyrene latex beads, pure bacterial samples and mixtures of bacterial species including Escherichia coli, Serratia marcescens, Pseudomonas aeruginosa and Bacillus subtilis. A similar relationship was utilised for polystyrene latex as a calibration line to enable the concentration of particles in a suspension to be determined according to the level of DEP collection. The particle concentration of an unknown test sample was found to lie within the predicted concentration range determined on the basis of DEP collection. In addition, the predicted limits were found only to deviate between -6.2 and +6.9% from the mean particle concentration.

A hypothesis on the metabolism of glyceryl trinitrate in vascular endothelial cells.

BACKGROUND: Extensive research has been conducted regarding the mechanism of action of glyceryl trinitrate (GTN). It is currently believed that GTN undergoes a thiol-dependent metabolic pathway and releases its active metabolite, nitric oxide (NO) and/or S-nitrosothiols (R-SNO). This activates guanylyl cyclase (GC) leading to the formation of cGMP, which is responsible for the relaxation of vascular smooth muscles and the inhibition of platelet aggregation. The lack of knowledge as to the precise mechanism of GTN action and the modulation of its formation has limited the prevention of tolerance to GTN. RESULTS: With cultured human vascular endothelial cells (EC), we showed that nitrite was first formed in endothelial cells whose concentration was dependent on reduced thiols. Cells preexposed to GTN significantly decreased the production of nitrite compared with cells that were not preexposed. Furthermore, we showed that thiols in cultured cells were oxidized during interaction with GTN, which correlated with the time of exposure to GTN. CONCLUSION: Nitrite is the first active intermediate of GTN metabolism in endothelial cells. The analysis of the changes of the blood nitrite and reduced thiols concentration is helpful for evaluating the vasodilatation activity of GTN during therapeutic treatments.

Dielectrophoretic analysis of microbes in water.

Traditional microbiological methods are still used extensively for analysis of micro-organisms in water. However, they are inefficient due to a high labour input requirement, a low sample capacity, and often a long time lag before results are available. Analytical stages involving incubation and growth (enrichments and colony isolation) contribute the greatest delay in reporting, although subsequent identification can also be protracted. The use of electrometric growth analysers (measuring impedance, conductance or capacitance changes) is now more common in water microbiology. Although these instruments can provide more rapid results and provide increased handling capacity, the bacterial generation times required to provide detectable changes cause delays and suitable selective media are not fully developed for all microbes of interest. Most other recent methods have equally disappointing drawbacks and thus extensive research continues in order to realise the ambition of 'real-time' analytical microbiology. Several research groups have demonstrated the potential of dielectrophoresis in providing microbial concentration, separation and identification systems which are not limited by bacterial growth and are therefore extremely rapid. Dielectrophoresis occurs when cells are placed in non-uniform electric fields. The cells move towards the electrodes (regardless of the direction of the applied field) as determined by their dielectric properties (conductivity and permittivity) rather than by their charge as occurs in electrophoresis. Also, the polarisability of the cells, and therefore the polarity and magnitude of the dielectrophoretic force, varies as a function of the electric field frequency. Because the dielectric properties of a particular cell type have characteristic frequency-dependent components, if cell collection at electrodes is observed across a frequency range, the collection spectrum produced is distinctive for the cell type under investigation. This can be exploited for analytical and separation applications in microbiology. This paper will describe rapid analytical techniques based on electrokinetic phenomena under research and development at York. These include dielectrophoretic enrichment, concentration and characterisation systems for the analysis of water bacteria and protozoa.

Liver organelle changes on exposure to hypoxia.

Exposure of mice to hypoxia reduces the length of each element of endoplasmic reticulum. This may shorten the path length of oxygen within the cell and have an adaptive advantage. There were no significant changes in the other organelles.

Gentisate, a salicylate metabolite with antioxidant properties.

Concentrations of gentisate found in plasma and synovial fluid were in excess of concentrations required to exert an antioxidant effect in vitro. Studies of diphenol/aminophenols suggested that an ortho or para configuration of functional groups was required for this in vitro antioxidant activity. Animal studies indicated some correlations between in vitro activity and pharmacology of these agents.

Nitrite May Be an Important EDRF Modulator.

Recently many studies suggested that S-nitrosothiols instead of active nitric oxide are EDRF. The studies showed that reduced thiols could not react directly with.NO at physiological pH (7.4) to form S-nitrosothiols but oxidized to nitrite which is very stable at pH 7.4. In contrast at low pH nitrite reacted rapidly with reduced thiols to form S-nitrosothiols. Similarly both nitrite and.NO gas reacted with deoxyHb and oxyHb at low pH but not at pH 7.4 to form HbFeII-NO complex or metHb and nitrate. The pH is a key factor to control this nitrosation process which is determined in vivo by oxygen tension and redox status. From these results a hypothesis is proposed that nitrite may be an EDRF modulator which controls the nitrosation level of reduced thiols in biological system.