Synthesis of paf-acether by E. coli K12

Paf-acether (platelet-activating factor) is one of the most potent mediator of inflammation released from and acting on most cells that participate in inflammatory diseases. Its molecular structure is 1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphocholine. Two metabolic steps are involved in its biosynthesis: the action of a phospholipase A2 on choline-containing membrane alkyl-ether lipids results in the production of lyso paf-acether and acetylation of the lyso compound by an acetyltransferase yields the biologically active molecule. Membrane alkyl-ether lipids can therefore be considered as potential precursors of paf-acether and their composition has been studied in various cell types. In this work, we investigated the presence of paf-acether in E. coli. Our results showed that paf-acether can be obtained from E. coli K12 under a variety of bacterial growth conditions. Paf-acether from E. coli exhibited the same physicochemical and biological characteristics as synthetic paf-acether and that from eucaryotic cells. Therefore, it appears that E. coli itself has the ability of producing paf-acether, a result that could be of some importance with respect to the pathogenesis of Enterobacteria and the use of E. coli in the recombinant DNA technology.     

Biosynthesis of paf-acether. XVI. Acetyltransferase specificity determines composition of paf-acether molecular species in human polymorphonuclear neutrophils.

In human neutrophils, the velocity of the lyso paf-acether:acetyl-CoA acetyltransferase reaction was almost 2-fold higher in the presence of lyso paf-acether bearing a 16:0 alkyl chain at the sn-1 position of glycerol than in that of its 18:0 analog. The paf-acether produced from an equimolar mixture of the two substrates was a 5:1 mixture, respectively, of the 16:0 and 18:0 species. The ratio of 16:0/18:0 lyso paf-acether in microsomal fractions, as analyzed by gas chromatography, was close to 1, whereas the paf-acether formed in these fractions from endogenous phospholipids was nearly exclusively of the 16:0 form. We conclude that acetyltransferase possesses a higher affinity for 16:0 than for 18:0 lyso-PAF and thus might control the molecular composition of paf-acether synthesized by stimulated human polymorphonuclear neutrophils.     

Biosynthesis of paf-acether. X. Phorbol myristate acetate-induced paf-acether biosynthesis and acetyltransferase activation in human neutrophils

We tested the hypothesis that protein kinase C might play a role in the biosynthesis of platelet-activating factor (paf-acether) in human neutrophils. PMA but not its inactive analog 4-alpha-phorbol-12,13-didecanoate induced lyso paf-acether production, followed by acetyltransferase activation, leading to paf-acether synthesis and release. Moreover, PMA was twice as powerful compared to opsonized zymosan (OPZ). 1-Oleoyl-2-acetyl-glycerol also induced acetyltransferase activation and paf and lyso paf production. The paf-acether formed by PMA or OPZ stimulation was composed of alkyl chains C16:0 (84.3 +/- 5% and 80.7 +/- 3.5%, respectively, and C18:0 (15.7 +/- 5% and 19.3 +/- 3.5%, respectively, means +/- SEM) as assessed by gas chromatography-electron capture detection. The inhibitor of protein kinase C, D-sphingosine, markedly decreased paf and lyso paf production and acetyltransferase activation in PMA- as well as OPZ-stimulated neutrophils. These results strongly suggest the involvement of protein kinase C in signal transduction during cell stimulation, leading to the paf biosynthesis.     

Immunoregulatory functions of paf-acether. I. Effect of paf-acether on CD4+ cell proliferation

Paf-acether or platelet-activating factor (1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a phospholipid mediator of inflammation initially described as a potent platelet-aggregating compound. It is newly formed by a variety of cells including monocytes and is now recognized as a major mediator of cell-cell interactions. The present studies were undertaken to determine whether paf-acether could modulate T cell function. We found that addition of paf-acether to CD4+ cells cultured with phytohemagglutinin markedly inhibited the proliferative response in a dose-dependent manner. Maximal inhibition occurred when paf-acether was present during the first 24 hr of cell culture and the presence of paf-acether did not alter the kinetics of CD4+ cell proliferation. Importantly, the mechanism by which paf-acether inhibited the proliferative response was not related to inhibition of interleukin 2 (IL-2) secretion since the amount of IL-2 in cultures was not altered and addition of exogenous IL-2 failed to restore the CD4+ cell proliferative response. Further, as judged by indirect immunofluorescence, paf-acether did not inhibit IL-2 receptor expression. Taken together, these data indicate that paf-acether interferes with some processes leading to CD4+ cell proliferation. This new role for the chemically defined monokine paf-acether emphasizes the potential role of inflammatory lipid mediators in the regulation of T cell response.     

Presence paf-acether mediator of inflammation in stools of patients with inflammatory enterocolitis

Paf-acether (platelet-activating factor, paf) is one of the most potent inflammatory mediators synthesised by and acting on most inflammatory cells. Paf causes gastric ulcerations and ischemic bowel necrosis. Recently, we described the presence of paf in stools of patients bearing ileal pouch-anal anastomosis with pouchitis. These findings prompted us to search for the presence of paf in stools of patients suffering from inflammatory bowel diseases: Crohn's disease and ulcerative colitis. The fraction corresponding to paf isolated from stools exhibited the same physicochemical and biological characteristics as synthetic paf. Reverse-phase high performance liquid chromatography revealed 80% of C16 and 20% of C18 paf. The presence of a biologically active phospholipid in human stools may bring new perspectives with respect to the study of gastrointestinal diseases as well as the use of paf antagonists in the treatment of inflammatory bowel diseases.     

Altering textural properties of fermented milk by using surface‐engineered Lactococcus lactis

Lactic acid bacteria are widely used for the fermentation of dairy products. While bacterial acidification rates, proteolytic activity and the production of exopolysaccharides are known to influence textural properties of fermented milk products, little is known about the role of the microbial surface on microbe–matrix interactions in dairy products. To investigate how alterations of the bacterial cell surface affect fermented milk properties, 25 isogenic Lactococcus lactis strains that differed with respect to surface charge, hydrophobicity, cell chaining, cell‐clumping, attachment to milk proteins, pili expression and EPS production were used to produce fermented milk. We show that overexpression of pili increases surface hydrophobicity of various strains from 3–19% to 94–99%. A profound effect of different cell surface properties was an altered spatial distribution of the cells in the fermented product. Aggregated cells tightly fill the cavities of the protein matrix, while chaining cells seem to be localized randomly. A positive correlation was found between pili overexpression and viscosity and gel hardness of fermented milk. Gel hardness also positively correlated with clumping of cells in the fermented milk. Viscosity of fermented milk was also higher when it was produced with cells with a chaining phenotype or with cells that overexpress exopolysaccharides. Our results show that alteration of cell surface morphology affects textural parameters of fermented milk and cell localization in the product. This is indicative of a cell surface‐dependent potential of bacterial cells as structure elements in fermented foods.     

Biosynthesis of paf-acether. XVII. Regulation by the CoA-independent transacylase in human neutrophils

Treatment of intact human polymorphonuclear neutrophils (PMN) with low concentrations of phorbol myristate acetate (PMA, 1-10 ng/ml) induced paf-acether (paf) and lyso paf formation, arachidonate release, and simultaneous inhibition of CoA-independent lyso paf: transacylase as assayed in a cell-free system. Inhibition of [3H]lyso paf reacylation was also observed when it was exogenously added to the PMA-treated intact PMN. When higher concentrations of PMA (40-100 ng/ml) were used, paf biosynthesis was severely impaired and the level of the CoA-independent transacylase activity returned to basal level. Since lyso paf appears to be the substrate for PMA-activated paf formation (remodeling pathway), we showed that [14C]acetate was incorporated into the paf molecule. By contrast, labeling with [3H]choline was not appropriate in this model. The presented results are against the involvement of a de novo route in paf synthesis initiated by PMA and open a new possibility of an important role for the CoA-independent transacylase in controlling the level of lyso paf availability for paf formation.     

Reconstitution by a butyrolactone autoregulator of the parental protein pattern in an asporogenous mutant of Streptomyces griseus

Abstract The lipoteichoic acid (LTA) isolated from Streptococcus cremoris T⁵, a representative strain from the fermented milk product 'viili', is shown by crossed immunoelectrophoresis (CIE) to appear mainly in acylated form. Isolated plasma membranes contain two LTA antigens, as revealed by co-CIE with isolated LTA. It is concluded from immunoadsorption experiments with washed intact cells that LTA from the membrane structure is also detectable on the surface of intact cells. The significance of these findings for the quality of thick or slimy fermented milk products is discussed.     

Changes in peptic digestibility of bovine beta-lactoglobulin as a result of food processing studied by capillary electrophoresis and immunochemical methods

Digestion studies constitute a functional tool for allergen characterisation. This strategy for investigating allergenic proteins relates to the observation of increased proteolytic resistance of some proteins recognised to exhibit allergenic potential. beta-Lactoglobulin (betaLG) is one of the major whey proteins, a potent milk allergen and shows a high stability against peptic hydrolysis in its native form. In order to study the impact of milk fermentation process on its digestibility, two complementary analytical methods were applied: capillary zone electrophoresis (CZE) to quantitatively study proteolytic degradation of betaLG isolated from different fermented bovine milk products, and enzyme linked immunosorbent assay (ELISA) to assess differences in immunoreactivity. betaLG, isolated from either raw or pasteurised cow's milk (CM), as expected, showed only minimal digestibility (less than 10% in 2 h). However, when raw milk or pasteurised milk was fermented, the rate of peptic digestion of the protein significantly increased (up to 45% in 2 h). In accordance with changes in digestibility, the immunochemical response for all fermented samples was lower than that of non-fermented references. Raw and pasteurised milk "naturally" fermented in our laboratory only resulted in a slight reduction (betaLG detected is still in the range of milligrams per gram sample), whereas the industrially manufactured sour milk as well as the "Acidophilus milk" reflected a remarkably lower level of immunoreactivity (55-56 microg/g sample).     

Antimutagenicity of milk fermented byEnterococcus fœcium

The diethyl ether extracts isolated from unfermented milk and milk fermented byEnterococcus fœcium exhibited dose-dependent inhibition of mutagenesis induced by N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), nitrovin (NIT), 5-nitro-2-furylacrylic acid (NFA) and UV-irradiation on the Ames bacterial test (Salmonella typhimurium strains TA97 and TA100) and the unicellular flagellateEuglena gracilis. Overall, the fermented milk extract was the most active against UV-irradiation, less active against NIT and MNNG, and the least active against NFA on bacteria. The highest antibleaching effects were observed against MNNG. The differences between antimutagenic effects from fermented and unfermented milk extracts were determined to be statistically significant at the 0.95 CI level.     

Leukocytes utilize myeloperoxidase-generated nitrating intermediates as physiological catalysts for the generation of biologically active oxidized lipids and sterols in serum

The initiation of lipid peroxidation and the concomitant formation of biologically active oxidized lipids and sterols is believed to play a central role in the pathogenesis of inflammatory and vascular disorders. Here we explore the role of neutrophil- and myeloperoxidase (MPO)-generated nitrating intermediates as a physiological catalyst for the initiation of lipid peroxidation and the formation of biologically active oxidized lipids and sterols. Activation of human neutrophils in media containing physiologically relevant levels of nitrite (NO(2)(-)), a major end product of nitric oxide (nitrogen monoxide, NO) metabolism, generated an oxidant capable of initiating peroxidation of lipids. Formation of hydroxy- and hydroperoxyoctadecadienoic acids [H(P)ODEs], hydroxy- and hydroperoxyeicosatetraenoic acids [H(P)ETEs], F(2)-isoprostanes, and a variety of oxysterols was confirmed using on-line reverse phase HPLC tandem mass spectrometry (LC/MS/MS). Lipid oxidation by neutrophils required cell activation and NO(2)(-), occurred in the presence of metal chelators and superoxide dismutase, and was inhibited by catalase, heme poisons, and free radical scavengers. LC/MS/MS studies demonstrated formation of additional biologically active lipid and sterol oxidation products known to be enriched in vascular lesions, such as 1-hexadecanoyl-2-oxovalaryl-sn-glycero-3-phosphocholine, which induces upregulation of endothelial cell adhesion and chemoattractant proteins, and 5-cholesten-3beta-ol 7beta-hydroperoxide, a potent cytotoxic oxysterol. In contrast to the oxidant formed during free metal ion-catalyzed reactions, the oxidant formed during MPO-catalyzed oxidation of NO(2)(-) readily promoted lipid peroxidation in the presence of serum constituents. Collectively, these results suggest that phagocytes may employ MPO-generated reactive nitrogen intermediates as a physiological pathway for initiating lipid peroxidation and forming biologically active lipid and sterol oxidation products in vivo.     

Biosynthesis of paf-acether. XI. Regulation of acetyltransferase by enzyme-substrate imbalance

Acetyl-CoA:1-O-alkyl-sn-glycero-3-phosphocholine acetyltransferase is the key enzyme in paf-acether (paf) biosynthesis, since it yields the active mediator from its nonacetylated precursor, lyso-paf. In microsomal fractions obtained from the ionophore A23187-stimulated human polymorphonuclear neutrophils, the optimal conditions allowing the full acetylation of lyso-paf were: 2-2.5 mg.ml-1 bovine serum albumin, 40 microM lyso-paf, 200 microM acetyl-CoA and acetyltransferase of high specific activity, at least 18 nmol.min-1.mg protein- -1. The reaction frequently stopped before the substrate was consumed due to spontaneous decay of the enzyme activity at 37 degrees C and inhibition of the enzyme by the paf formed in the reaction. However, low concentrations of acetyltransferase substrates (lyso-paf or lysophosphatidylcholine) and the antioxidant dithiothreitol, but not the inhibitors of proteinases or phosphatases, protected the enzyme against decay. In contrast, high concentrations of those lyso substrates inhibited the enzyme activity in the assay. This inhibition as well as that due to paf was overcome by raising the concentration of the enzyme contained in the microsomal fraction or the bovine serum albumin in the assay. These results suggest that the biosynthesis of paf in cell-free assay and most probably in intact cells might be controlled to a larger extent by the acetyltransferase concentration rather than by that of its substrates.     

The release of bradykinin in bovine mastitis.

The kinin peptides are released during inflammation and are amongst the most potent known mediators of vasodilatation, pain and oedema. Despite early reports of the presence of kinins in milk, no previous study has investigated the role of the kinin system in bovine mastitis. The present study indicated that mastitis was accompanied by raised levels of bradykinin (BK) in milk and the increased levels of BK correlated with the severity of mastitis. Raised BK levels in mastitic milk were not dependent on the presence of inflammatory cells, nor were they secondary to changes in blood levels of BK. In milk from sub-clinically inflamed quarters, BK was raised in those milks where Staphylococcus aureus (S. aureus) was isolated but not in those milks where no pathogen was isolated. Increasing S. aureus artificially, also caused an increase in the milk BK. Increases in milk BK were not restricted only to the mastitic quarters of the udder. In udders in which mastitis was detected in one or more quarters, BK increases were also detected in the apparently uninvolved quarters.     

Nucleases isolated from Chelidonium majus L. milky sap can induce apoptosis in human cervical carcinoma HeLa cells but not in Chinese Hamster Ovary CHO cells

Milky sap isolated from Chelidonium majus L. (Greater Celandine) serves as a rich source of various biologically active substances such as alkaloids, flavonoids and phenolic acids. Previous research showed that the activity of Ch. majus milky sap may depend also on the presence of biologically active proteins. The goal of this study was to evaluate the biological effect of two nucleases isolated from Ch. majus milk sap, CMN1 of 20 kDa and CMN2 of 36 kDa, on HeLa and CHO tumour cell lines. Both studied nucleases together with other proteins in the sap of the plant are involved in stress and defence reactions against different pathogens. After 48 h incubation of CMN1 and CMN2 only with HeLa cells, the dependence between the number of apoptotic lesions and the concentration of applied nuclease was observed. The highest proapoptotic activity was induced by 13.3 ng/ml concentration of CMN2 collected in May (62 +/- 3% HeLa cells were apoptotic). Moreover, the proportion of necrotic cells in all concentrations of the nucleases and both cell lines was relatively low (1-8 +/- 0.5%). In summary, results of this study show that purified nucleases CMN1 and CMN2 isolated from Ch. majus milky sap exhibit apoptotic activity in HeLa tumour cell line, but not in CHO cells, without inflammatory reaction.     

Oxidized phospholipids as modulators of inflammation in atherosclerosis

PURPOSE OF REVIEW: This review will summarize recent evidence demonstrating that biologically active phospholipid oxidation products modulate inflammatory reactions. RECENT FINDINGS: Structural identification of new biologically active oxidized phospholipids and the finding that they can also be formed at inflammatory sites other than the atherosclerotic lesion have expanded the potential role of these compounds in inflammation beyond atherogenesis. Various signaling pathways are induced by oxidized phospholipids, leading to the expression of inflammatory genes by mechanisms that differ from those mediated by the classic inflammatory agonists tumor necrosis factor or lipopolysaccharide. Furthermore, oxidized phospholipids can bind to pattern recognition molecules and thus potently influence inflammation and immune responses during host defense. SUMMARY: During inflammatory processes biologically active lipid oxidation products accumulate that modulate the inflammatory process and may determine the fate and outcome of the body's reaction in acute inflammation during host defense. Oxidized phospholipids may induce and propagate chronic inflammatory processes; however, evidence is accumulating that cells and tissues respond towards these oxidatively formed stress signals also by activation of anti-inflammatory, cytoprotective reactions.     

Popular ovine and caprine fermented milks

Ovine and caprine milk are widely produced in semi-arid countries, and mainly utilised for milk consumption and the manufacture of a wide range of cheeses, fermented milk products (e.g. liquid, viscous, concentrated and dried) and to a lesser degree milk powder. The primary aim of this review is to provide a critical analysis of the main components of milk from different ovine and caprine breeds for the manufacture of fermented milk products, i.e. traditional and industrial. This paper reviews the latest scientific developments in fermented milks, including probiotic products, and recommendations to improve the characteristics of the final product(s). Although ovine milk is successfully used to manufacture many fermented milk products, the solids content of milk from certain breeds of caprine needs to be fortified to enhance the protein content and modify the processing conditions to ensure products acceptable to the consumer.     

Identification of dipeptidyl peptidase III in human neutrophils

We have found activity of dipeptidyl peptidase (DPP) III, one of the most important enkephalin-degrading enzymes in the central nervous system, in human neutrophils. HPLC analysis of the peptide fragments produced by treatment of leucine-enkephalin with isolated neutrophils in the presence of inhibitors of other enkephalin-degrading enzymes revealed that the enzyme in human neutrophils cleaved dipeptides from the NH(2) terminus of leucine-enkephalin, suggesting the presence of DPPIII activity in human neutrophils. Using a specific synthesized substrate and proteinase inhibitors, it was found that the neutrophils have 19.2 +/- 3.6 microM/h/5 x 10(6) cells of beta-naphthylamine for the enzyme. It was also confirmed that spinorphin and tynorphin, both reported to inhibit the activities of enkephalin-degrading enzymes, had potent inhibitory activities (IC(50): 4.0 and 0.029 microg/ml, respectively) against the enzyme. The presence of DPPIII activity in human neutrophils suggests that the biologically active peptides which are associated with enkephalin play a physiological role in regulating enkephalin or inflammatory mechanisms in peripheral tissues.     

Dominant lactic acid bacteria and their technological properties isolated from the Himalayan ethnic fermented milk products

Ethnic people of the Himalayan regions of India, Nepal, Bhutan and China consume a variety of indigenous fermented milk products made from cows milk as well as yaks milk. These lesser-known ethnic fermented foods are dahi, mohi, chhurpi, somar, philu and shyow. The population of lactic acid bacteria (LAB) ranged from 10(7) to 10(8) cfu/g in these Himalayan milk products. A total of 128 isolates of LAB were isolated from 58 samples of ethnic fermented milk products collected from different places of India, Nepal and Bhutan. Based on phenotypic characterization including API sugar test, the dominant lactic acid bacteria were identified as Lactobacillus bifermentans, Lactobacillus paracasei subsp. pseudoplantarum, Lactobacillus kefir, Lactobacillus hilgardii, Lactobacillus alimentarius, Lactobacillus paracasei subsp. paracasei, Lactobacillus plantarum, Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris and Enterococcus faecium. LAB produced a wide spectrum of enzymes and showed high galactosidase, leucine-arylamidase and phosphatase activities. They showed antagonistic properties against selected Gram-negative bacteria. None of the strains produced bacteriocin and biogenic amines under the test conditions used. Most strains of LAB coagulated skim milk with a moderate drop in pH. Some strains of LAB showed a high degree of hydrophobicity, suggesting these strains may have useful adhesive potential. This paper is the first report on functional lactic acid bacterial composition in some lesser-known ethnic fermented milk products of the Himalayas.     

Arachidonic acid metabolism as a potential mediator of cardiac fibrosis associated with inflammation

An increase in left ventricular collagen (cardiac fibrosis) is a detrimental process that adversely affects heart function. Strong evidence implicates the infiltration of inflammatory cells as a critical part of the process resulting in cardiac fibrosis. Inflammatory cells are capable of releasing arachidonic acid, which may be further metabolized by cyclooxygenase, lipoxygenase, and cytochrome P450 monooxygenase enzymes to biologically active products, including PGs, leukotrienes, epoxyeicosatrienoic acids, and hydroxyeicosatetraenoic acids. Some of these products have profibrotic properties and may represent a pathway by which inflammatory cells initiate and mediate the development of cardiac fibrosis. In this study, we critically review the current literature on the potential link between this pathway and cardiac fibrosis.