The involvement of the proteinase of Streptococcus faecium ATCC 9790 in the stimulation of its autolysin activity by dodecylglycerol

Treatment of Streptococcus faecium ATCC 9790 with 3.5 micrograms/ml of dodecylglycerol produces a nonwall entity found in the 25,000 X g supernatant cell fraction which activates the autolysin activity of S. faecium. The stimulation of the autolysin activity by dodecylglycerol mimics the activation of the autolysin from a latent to an active form by trypsin and other proteolytic enzymes. This stimulation of autolytic activity by dodecylglycerol can be reversed by specific proteinase inhibitors. Dodecylglycerol also markedly stimulates the proteinase activity endogenous to S. faecium, and this stimulation can be reversed by several proteinase inhibitors. It is concluded that one primary antibacterial mode of action of dodecylglycerol is to stimulate the proteinase of S. faecium which activates the cell's autolysin and thereby prevents bacterial growth.     

Induction of autolysis in Streptococcus faecium

Autolysis of exponential-phase Streptococcus faecium cells was promoted by pretreating the bacteria (freezing-thawing; -70 degrees C) in Tris buffer, followed by incubation at 37 degrees C in the same buffer. The effect was dependent on Tris concentration. The pretreatment provoked ultrastructurally visible damage with extensive loss of K+ and leakage of UV-absorbing components. No autolysis was observed when the bacteria frozen-thawed in Tris were incubated in the presence of the autolysin inhibitor N-bromosuccinimide nor when they had been grown in the presence of chloramphenicol or tetracycline. Furthermore, two autolytic-defective mutants, EC31 and EC78, isolated from S. faecium, did not autolyse when frozen-thawed and incubated in Tris. Freezing-thawing in Tris, however, imparted extensive cell damage to the mutants and to the antibiotic-treated bacteria as well as considerable leakage of K+ and UV-absorbing materials. These observations indicate that the lysis of S. faecium reported above is due to the activity of the endogenous bacterial autolysin. Induction of autolysis of S. faecium by freezing-thawing was also observed, although to a lesser extent, when Tris was replaced by imidazole.     

Antibacterial activity of extracts and neolignans from Piper regnellii (Miq.) C. DC. var. pallescens (C. DC.) Yunck

The evaluation of the activity of the aqueous and ethyl acetate extracts of the leaves of Piper regnellii was tested against gram-positive and gram-negative bacteria. The aqueous extract displayed a weak activity against Staphylococcus aureus and Bacillus subtilis with minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of 1000 micrograms/ml. The ethyl acetate extract presented a good activity against S. aureus and B. subtilis with MIC and MBC at 15.62 micrograms/ml. In contrast to the relative low MICs for gram-positive bacteria, gram-negative bacteria were not inhibited by the extracts at concentrations < or = 1000 mg/ml. The ethyl acetate extract was fractionated on silica gel into nine fractions. The hexane and chloroform fractions were active against S. aureus (MIC at 3.9 micrograms/ml) and B. subtilis (MIC at 3.9 and 7.8 micrograms/ml, respectively). Using bioactivity-directed fractionation, the hexane fraction was rechromatographed to yield the antimicrobial compounds 1, 2, 5, and 6 identified as eupomatenoid-6, eupomatenoid-5, eupomatenoid-3, and conocarpan, respectively. The pure compounds 1 and 2 showed a good activity against S. aureus with MIC of 1.56 micrograms/ml and 3.12 micrograms/ml, respectively. Both compounds presented MIC of 3.12 micrograms/ml against B. subtilis. The pure compound 6 named as conocarpan was quite active against S. aureus and B. subtilis with MIC of 6.25 micrograms/ml. The antibacterial properties of P. regnellii justify its use in traditional medicine for the treatment of wounds, contaminated through bacteria infections.     

Antibacterial activity of ethanolic and aqueous extracts of Acacia aroma Gill. ex Hook et Arn

The purpose of the present study was to investigate the antibacterial activity of seven ethanolic extracts and three aqueous extracts from various parts (leaves, stems and flowers) of A. aroma against 163 strains of antibiotic multi-resistant bacteria. The disc diffusion assay was performed to evaluate antibacterial activity of the A. aroma crude extracts, against several Gram-positive bacteria (E. faecalis, S. aureus, coagulase-negative stahylococci, S. pyogenes, S. agalactiae, S. aureus ATCC 29213, E. faecalis ATCC 29212) and Gram-negative bacteria (E. coli., K. pneumoniae, P. mirabilis, E. cloacae, S. marcescens, M morganii, A. baumannii, P. aeruginosa, S. maltophilia, E. coli ATCC 35218, P. aeruginosa ATCC 27853, E. coli ATCC 25922). All ethanolic extracts showed activity against gram-positive bacteria. Among all obtained extracts, only leaf and flower fluid extracts showed activity against Gram-negative bacteria. Based on this bioassay, leaf fluid extracts tended to be the most potent, followed by flower fluid extracts. Minimal inhibitory concentration (MIC) values of extracts and antibiotics were comparatively determined by agar and broth dilution methods. Both extracts were active against S. aureus, coagulase-negative stahylococci, E. faecalis and E. faecium and all tested Gram-negative bacteria with MIC values from 0.067 to 0.308 mg/ml. In this study the minimal bactericidal concentration (MBC) values were identical or twice as high than the corresponding MIC for leaf extracts and four or eight times higher than MIC values for flower extracts. This may indicate a bactericidal effect. Stored extracts have similar antibacterial activity as recently obtained extracts. The A. aroma extracts of leaves and flowers may be useful as antibacterial agents against Gram- negative and Gram-positive antibiotic multi-resistant microorganisms.     

Phosphatidylcholine stimulates the activity of UDP-Gal beta 1-4 galactosyltransferase in normal human kidney proximal tumour cells

Effects of various lipid components of low density lipoproteins (LDL) and serine on the regulation of UDP-Gal-beta 1-4-galactosyltransferase (GalT-2) activity have been investigated in normal proximal tubular (PT) cells. Addition of exogenous serine (0.1-0.75 mM), cholesterol (0-200 micrograms/ml medium), linoleic acid and oleic acid (0.1-0.75 mM) for 4 hr at 37 degrees C did not suppress the activity of GalT-2 in PT cells. Similarly, incubation of cells with glucosylceramide and lactosylceramide (25-50 micrograms/ml medium) did not alter GalT-2 activity in cells as compared to control. In contrast, palmitic acid (0-0.75 mM), phosphatidylethanolamine and sphingomyelin (0-200 micrograms/ml) stimulated GalT-2 activity by 20-36% as compared to control. Incubation of PT cells with D-alpha-dipalmitoyl phosphatidylcholine (0-200 micrograms/ml medium) also stimulated the activity of GalT-2, maximum stimulation (200%) occurring with 25 micrograms phosphatidylcholine/ml medium. However, at a higher concentration (200 micrograms/ml), the stimulation of the activity of GalT-2 was in the order of 27% compared to control. Dioleylphosphatidylcholine did not alter GalT-2 activity in PT cells. Thus, it is concluded that (i) various lipid components, sphingosine and serine present in LDL are not involved in the LDL-mediated suppression of GalT-2 activity in normal PT cells, and (ii) stringent structural requirements in the phosphatidylcholine molecule are necessary to exert a time and concentration dependent stimulation of GalT-2 activity.     

Effect of Lactic Acid Bacteria on Growth of Staphylococcus aureus

Cultures of lactic acid bacteria, mostly from foods, were tested for their effect on the growth of Staphylococcus aureus in Trypticase Soy Broth (BBL). Some of the effectors, e.g., Streptococcus faecalis, S. faecium, Lactobacillus lactis, L. brevis, and Leuconostoc mesenteroides, stimulated growth of S. aureus during early hours of growth, especially at higher temperatures of incubation, but most cultures were inhibitory, and some (S. faecium and L. mesenteroides) were even killing by the time of attainment of the maximal phase of growth of the Staphylococcus. Low-temperature meat lactobacilli and Leuconostoc dextranicum inhibited S. aureus at 10, 15, 20, and 25 C throughout its growth. Streptococcus faecalis var. liquefaciens inhibited at these temperatures and at 30 and 37 C, as well. When the ratio of effectors to staphylococci in the inoculum was 100:1, the three enterococci, the meat Lactobacillus, and L. dextranicum prevented the attainment of 5 x 10(6) staphylococci per milliliter at 15 C, and all but the meat Lactobacillus did so at 22 C. A ratio of 1:1 accomplished similar results at 15 C, except that S. aureus was only delayed for 12 hr by S. faecalis. A ratio of 1:100 usually was ineffective. In general, the more effector bacteria there were in the inoculum, the greater was the overall inhibition (or stimulation) of S. aureus. Inhibition was most effective at 10 or 15 C, less so at 20 or 25 C, and least at 30 or 37 C, whereas stimulation during early growth was greater at the higher temperatures. Results with different strains of the effectors and with two strains of S. aureus were similar, for the most part.     

Identification of a streptococcal penicillin-binding protein that reacts very slowly with penicillin.

Penicillin-binding protein (PBP) 5 of Streptococcus faecium ATCC 9790 has an unusually low affinity for penicillin (50% binding occurred at a penicillin level of 8 micrograms/ml after 60 min of incubation, and the protein only became labeled after 20 min of incubation with high concentrations of radioactive penicillin). PBPs with similar properties are carried by strains of Streptococcus durans, Streptococcus faecalis, and Streptococcus lactis but not by strains of groups A, B, C, and G streptococci or Streptococcus pneumoniae. The strains carrying the slow-reacting PBP demonstrated a sensitivity to penicillin that was several hundred times lower than that of strains not carrying it. Spontaneous mutants with minimal inhibitory concentrations of penicillin of 20, 40, and 80 micrograms/ml were isolated from S. faecium ATCC 9790. They all showed a dramatic increase in the amount of slow-reacting PBP produced. Mutants with increased penicillin resistance were also isolated from wild-type strains of S. durans, S. faecalis, and S. faecium. All of them carried a greater amount of the slow-reacting PBP than that carried by the parent. Finally, it was found that resistant S. faecium ATCC 9790 mutants grew normally in the presence of penicillin concentrations that were far above that saturating all PBPs except PBP 5. Cell growth was, on the contrary, inhibited by a penicillin concentration that saturated the slow-reacting PBP by 90%. This penicillin dose was equal to the minimal inhibitory concentration.     

Inhibition of Listeria monocytogenes by fatty acids and monoglycerides.

Fatty acids and monoglycerides were evaluated in brain heart infusion broth and in milk for antimicrobial activity against the Scott A strain of Listeria monocytogenes. C12:0, C18:3, and glyceryl monolaurate (monolaurin) had the strongest activity in brain heart infusion broth and were bactericidal at 10 to 20 micrograms/ml, whereas potassium (K)-conjugated linoleic acids and C18:2 were bactericidal at 50 to 200 micrograms/ml. C14:0, C16:0, C18:0, C18:1, glyceryl monomyristate, and glyceryl monopalmitate were not inhibitory at 200 micrograms/ml. The bactericidal activity in brain heart infusion broth was higher at pH 5 than at pH 6. In whole milk and skim milk, K-conjugated linoleic acid was bacteriostatic and prolonged the lag phase especially at 4 degrees C. Monolaurin inactivated L. monocytogenes in skim milk at 4 degrees C, but was less inhibitory at 23 degrees C. Monolaurin did not inhibit L. monocytogenes in whole milk because of the higher fat content. Other fatty acids tested were not effective in whole or skim milk. Our results suggest that K-conjugated linoleic acids or monolaurin could be used as an inhibitory agent against L. monocytogenes in dairy foods.     

Inhibition of Listeria monocytogenes by fatty acids and monoglycerides.

Fatty acids and monoglycerides were evaluated in brain heart infusion broth and in milk for antimicrobial activity against the Scott A strain of Listeria monocytogenes. C12:0, C18:3, and glyceryl monolaurate (monolaurin) had the strongest activity in brain heart infusion broth and were bactericidal at 10 to 20 micrograms/ml, whereas potassium (K)-conjugated linoleic acids and C18:2 were bactericidal at 50 to 200 micrograms/ml. C14:0, C16:0, C18:0, C18:1, glyceryl monomyristate, and glyceryl monopalmitate were not inhibitory at 200 micrograms/ml. The bactericidal activity in brain heart infusion broth was higher at pH 5 than at pH 6. In whole milk and skim milk, K-conjugated linoleic acid was bacteriostatic and prolonged the lag phase especially at 4 degrees C. Monolaurin inactivated L. monocytogenes in skim milk at 4 degrees C, but was less inhibitory at 23 degrees C. Monolaurin did not inhibit L. monocytogenes in whole milk because of the higher fat content. Other fatty acids tested were not effective in whole or skim milk. Our results suggest that K-conjugated linoleic acids or monolaurin could be used as an inhibitory agent against L. monocytogenes in dairy foods.     

Common occurrence of plasmid DNA and vancomycin resistance in Leuconostoc spp.

Resistance to vancomycin permitted detection, in a culture of Streptococcus cremoris 290PC, of a contaminant gram-positive coccus. Morphological and physiological characteristics indicated that this bacterium was a strain of Leuconostoc sp., designated PO184. This strain contained four plasmid species, which were distinct from those harbored by S. cremoris 290PC. Antibiotic disk susceptibility tests indicated that Leuconostoc sp. strain PO184 was also resistant to sulfathiazole and trimethoprim and susceptible to 17 other antimicrobials. The MIC of vancomycin for this strain was greater than 2,000 micrograms/ml, and resistance did not depend on drug inactivation. Leuconostoc sp. strain PO184 produced a substance which was inhibitory to S. cremoris U134, but not to S. lactis ATCC 11454. Five other leuconostocs produced substances with antibacterial activity. Of 18 strains of Leuconostoc sp., 14 were resistant to at least 500 micrograms of vancomycin per ml, including four L. oenos strains which harbored no plasmid DNA in the 1- to 76-megadalton range. Twelve Leuconostoc sp. strains contained at least one plasmid species in this mass range. These findings are discussed from the physiological, taxonomical, and ecological standpoints and with regard to their potential applications.     

Antibacterial and bactericidal activities of tea extracts and catechins against methicillin resistant Staphylococcus aureus]

We examined tea extract, (-) epigallocatechin gallate (EGCg) and theaflavin digallate (TF3) for their antibacterial and bactericidal activities against methicillin resistant Staphylococcus aureus (MRSA) and food poisoning strains of S. aureus. Twenty percent tea extract (50 microliters), EGCg (63 micrograms) and TF3 (125 micrograms) added to one ml of culture medium each inhibited the growth of all strains of MRSA and food poisoning S. aureus tested. Tea extract showed also a bactericidal activity against MRSA even at the same concentration of as in ordinarily brewed tea. EGCg at a concentration of 250 micrograms/ml showed a bactericidal activity against MRSA but not against food poisoning S. aureus, but at 500 micrograms/ml reduced markedly the viable number within 48h. These results suggest that tea and catechin can be used as prophylactic agents against MRSA infection.     

Activation of peritoneal macrophages by orally administered ether analogues of lysophospholipids.

Oral administration of dodecylglycerol, inflammatory product of cancerous tissues, and the alkyl lysophospholipid derivative, 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3-choline), greatly activated mouse peritoneal macrophages. The activation was dose related and was assessed as increased Fc-mediated ingestion of red blood cells, superoxide production, chemiluminescence activity, and incorporation of radioactive thymidine and leucine. Furthermore, the data show that dodecylglycerol or ET-18-OCH3-choline was capable of inducing equally high levels of macrophage activation and cytotoxic action on tumor cells, just as occurs with intraperitoneal administration. Dodecylglycerol appeared to activate the macrophages at a relatively lower dose (5 micrograms/mouse) than ET-18-OCH3-choline (15 micrograms/mouse). The optimal oral doses required to activate macrophages for ingestion and cytotoxic activities were relatively higher than previously observed when these agents were administered intraperitoneally. Thus, the dose difference provided crucial information for correlating oral dosages with in vivo concentration of these agents as bioassayed by macrophage activation. These observations have extended and further support our earlier findings that these agents are effective immunopotentiators and thus could therapeutically be used to activate macrophages for cytotoxic effects on tumor cells via the oral route.     

Effects of monolaurin on ruminal methanogens and selected bacterial species from cattle, as determined with the rumen simulation technique

Before being able to implement effective ruminal methane mitigation strategies via feed supplementation, the assessment of side effects on ruminal fermentation and rumen microbial populations is indispensable. In this respect we investigated the effects of monolaurin, a methane-mitigating lipid, on methanogens and important carbohydrate-degrading bacteria present in ruminal fluid of dairy cattle in continuous culture employing the rumen simulation technique. In six experimental runs, each lasting for 10 days, four diets with different carbohydrate composition, based on hay, maize, wheat and a maize-wheat mixture, either remained non-supplemented or were supplemented with monolaurin and incubated in a ruminal-fluid buffer mixture. Incubation liquid samples from days 6 to 10 of incubation were analyzed with relative quantitative polymerase chain reaction (qPCR) of 16S rRNA genes to assess monolaurin-induced shifts in specific rumen microbial populations in relation to the corresponding non-supplemented diets. Monolaurin completely inhibited Fibrobacter succinogenes in all diets while the response of the other cellulolytic bacteria varied in dependence of the diet. Megasphaera elsdenii remained unaffected by monolaurin in the two diets containing maize, but was slightly stimulated by monolaurin with the wheat and largely with the hay diet. The supply of monolaurin suppressed Methanomicrobiales below the detection limit with all diets, whereas relative 16S rRNA gene copy numbers of Methanobacteriales increased by 7-fold with monolaurin in case of the hay diet. Total Archaea were decreased by up to over 90%, but this was significant only for the wheat containing diets. Thus, monolaurin exerted variable effects mediated by unknown mechanisms on important ruminal microbes involved in carbohydrate degradation, along with its suppression of methane formation. The applicability of monolaurin for methane mitigation in ruminants thus depends on the extent to which adverse effects on carbohydrate-degrading bacteria actually impair the supply of digested carbohydrates to the animal.     

Regulation of glycosphingolipid glycosyltransferase by low density lipoprotein receptors in cultured human proximal tubular cells

We have shown previously that low density lipoproteins (LDL) suppressed the synthesis of lactosylceramide in normal human proximal tubular cells, but stimulated such synthesis in proximal tubular cells from LDL receptor negative subjects (Chatterjee, S., Clarke, K., and Kwiterovich, P.O., Jr. (1986) J. Biol. Chem. 261, 13474-13479). To understand the mechanism(s) of this effect of LDL, we have studied here the effects of LDL on the activity of UDP-GalCer:beta-galactosyltransferase (GalT-2). Maximum suppression (70-80%) of the activity of GalT-2 in normal proximal tubular cells at 37 degrees C occurred at a LDL concentration of 25 micrograms/ml medium. Such suppression was not observed either when the cells were incubated with LDL at 4 degrees C, or when the cells were preincubated with leupeptin, followed by incubation with LDL at 37 degrees C. High density lipoproteins and fetuin did not suppress the activity of GalT-2 in normal proximal tubular cells. In contrast LDL modified by reductive methylation (M-LDL, 100 micrograms/ml) stimulated the activity of GalT-2, approximately 3-fold. The effects of LDL and M-LDL were not related to their glycosphingolipid content. Much less suppression and stimulation of the activity of GalT-2 in proximal tubular cells by LDL and M-LDL, respectively, was found in normal human skin fibroblasts, Chinese hamster ovary cells, and bovine smooth muscle cells, suggesting that the LDL-mediated effect may be tissue-specific. In cells grown to very high density, the activity of the LDL receptor is decreased, and there was less suppression of GalT-2 activity by LDL. In normal proximal tubular cells, LDL stimulated the activity of UDP-Gal:LacCer, alpha-galactosyltransferase activity, UDP-Gal:LcOse3Cer, beta-galactosyltransferase, and CMP-NeuAc:LacCer,alpha-sialyltransferase activity but did not alter the activity of sulfotransferase. In conclusion, LDL that entered the normal proximal tubular cells via the LDL receptor-mediated pathway decreased GalT-2 activity, an effect that was dependent upon the binding, internalization, and degradation of receptor-bound LDL. In contrast LDL that entered normal or LDL receptor-negative proximal tubular cells via an LDL receptor-independent pathway failed to suppress GalT-2 activity, and led to a stimulation of LacCer synthesis.     

Characterization of a bacteriocin produced by Enterococcus faecium GM-1 isolated from an infant

AIM: To partially characterize the bacteriocin produced by the GM-1 strain of Enterococcus faecium, isolated from the faeces of a newborn human infant. METHODS AND RESULTS: The bacteriocin produced by E. faecium GM-1 showed a broad spectrum of activity against indicator strains of Escherichia coli, Staphylococcus aureus, Vibrio spp., Salmonella typhimurium, Listeria monocytogenes, Lactobacillus acidophilus, and Streptococcus thermophilus. Treatment of the GM-1 bacteriocin with proteolytic enzymes reduced its inhibitory activities. The bacteriocin was stable at 100 degrees C for 20 min and displayed inhibitory activity at neutral pH. The optimal production of bacteriocin from E. faecium GM-1 was obtained when the culture conditions were pH 6.0-6.5 and 35-40 degrees C. The inhibitory activity of the bacteriocin was not substantially changed by the use of different carbon sources in the media, except when galactose was substituted for glucose. The use of a sole nitrogen source caused a decrease in inhibitory activity. A bacteriocin gene similar to enterocin P was identified from the total DNA of E. faecium GM-1 by PCR and direct sequencing methods. CONCLUSION: E. faecium GM-1, which was isolated from the faeces of a newborn baby, produces an enterocin P-like bacteriocin with inhibitory activity against Gram-positive and Gram-negative bacteria, including food-borne pathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: E. faecium GM-1, isolated from infant faeces, produces a new bacteriocin that is similar to enterocin P. This bacteriocin is heat stable and has a broad antibacterial spectrum that includes both Gram-positive and Gram-negative bacteria.     

Susceptibility and resistance of ruminal bacteria to antimicrobial feed additives

Susceptibility and resistance of ruminal bacterial species to avoparcin, narasin, salinomycin, thiopeptin, tylosin, virginiamycin, and two new ionophore antibiotics, RO22-6924/004 and RO21-6447/009, were determined. Generally, antimicrobial compounds were inhibitory to gram-positive bacteria and those bacteria that have gram-positive-like cell wall structure. MICs ranged from 0.09 to 24.0 micrograms/ml. Gram-negative bacteria were resistant at the highest concentration tested (48.0 micrograms/ml). On the basis of their fermentation products, ruminal bacteria that produce lactic acid, butyric acid, formic acid, or hydrogen were susceptible and bacteria that produce succinic acid or ferment lactic acid were resistant to the antimicrobial compounds. Selenomonas ruminantium was the only major lactic acid-producing bacteria resistant to all the antimicrobial compounds tested. Avoparcin and tylosin appeared to be less inhibitory (MIC greater than 6.0 micrograms/ml) than the other compounds to the two major lactic acid-producing bacteria, Streptococcus bovis and Lactobacillus sp. Ionophore compounds seemed to be more inhibitory (MIC, 0.09 to 1.50 micrograms/ml) than nonionophore compounds (MIC, 0.75 to 12.0 micrograms/ml) to the major butyric acid-producing bacteria. Treponema bryantii, an anaerobic rumen spirochete, was less sensitive to virginiamycin than to the other antimicrobial compounds. Ionophore compounds were generally bacteriostatic, and nonionophore compounds were bactericidal. The specific growth rate of Bacteroides ruminicola was reduced by all the antimicrobial compounds except avoparcin. The antibacterial spectra of the feed additives were remarkably similar, and it appears that MICs may not be good indicators of the potency of the compounds in altering ruminal fermentation characteristics.     

Susceptibility and resistance of ruminal bacteria to antimicrobial feed additives.

Susceptibility and resistance of ruminal bacterial species to avoparcin, narasin, salinomycin, thiopeptin, tylosin, virginiamycin, and two new ionophore antibiotics, RO22-6924/004 and RO21-6447/009, were determined. Generally, antimicrobial compounds were inhibitory to gram-positive bacteria and those bacteria that have gram-positive-like cell wall structure. MICs ranged from 0.09 to 24.0 micrograms/ml. Gram-negative bacteria were resistant at the highest concentration tested (48.0 micrograms/ml). On the basis of their fermentation products, ruminal bacteria that produce lactic acid, butyric acid, formic acid, or hydrogen were susceptible and bacteria that produce succinic acid or ferment lactic acid were resistant to the antimicrobial compounds. Selenomonas ruminantium was the only major lactic acid-producing bacteria resistant to all the antimicrobial compounds tested. Avoparcin and tylosin appeared to be less inhibitory (MIC greater than 6.0 micrograms/ml) than the other compounds to the two major lactic acid-producing bacteria, Streptococcus bovis and Lactobacillus sp. Ionophore compounds seemed to be more inhibitory (MIC, 0.09 to 1.50 micrograms/ml) than nonionophore compounds (MIC, 0.75 to 12.0 micrograms/ml) to the major butyric acid-producing bacteria. Treponema bryantii, an anaerobic rumen spirochete, was less sensitive to virginiamycin than to the other antimicrobial compounds. Ionophore compounds were generally bacteriostatic, and nonionophore compounds were bactericidal. The specific growth rate of Bacteroides ruminicola was reduced by all the antimicrobial compounds except avoparcin. The antibacterial spectra of the feed additives were remarkably similar, and it appears that MICs may not be good indicators of the potency of the compounds in altering ruminal fermentation characteristics.     

Production of two bacteriocins in various growth conditions produced by gram-positive bacteria isolated from chicken cecum

Lactobacillus plantarum CLP29 and Enterococcus faecium CLE34 isolated from the cecal contents of young broiler chicks were identified based on physiological and biochemical characteristics, and identification was confirmed by 16S rRNA sequencing. Both bacteria showed a broad range of inhibitory action against bacteria such as Salmonella and Escherichia coli and produced two peptides, plantaricin CLP29 and enterocin CLE34. Treatment with proteinase K, trypase, or benase resulted in the loss of activity of the two peptides, confirming their proteinaceous nature. The highest activity levels for both bacteria were recorded in de Man?- Rogosa?- Sharpe agar at pH?5.0, 6.0, and 7.0, at 37?°C. Carbon and nitrogen sources affected the antibacterial activities of the two bacteriocins in different combinations, which suggested that the antibacterial abilities of different bacteriocins produced in nutrient sources were various.     

Arachidonic acid metabolism of the murine eosinophil. II. Involvement of the lipoxygenase pathway in the response to the lymphokine eosinophil stimulation promoter

Eosinophil stimulation promoter (ESP) is a murine lymphokine that enhances the migration of eosinophils. Exogenous arachidonic acid between 0.5 and 2 micrograms/ml potentiated the activity of ESP on murine eosinophil migration, whereas such concentrations did not affect migration in the absence of ESP. Among the lipoxygenase products identified from an enriched population of murine eosinophils, leukotriene B4 (optimal activity at 100 ng/ml) and 12-HETE (optimal activity at 2 micrograms/ml) stimulated migration of these cells. Another lipoxygenase product from these cells 15-HETE inhibited ESP-induced migration; between 5 and 10 micrograms/ml 15-HETE decreased by one-half both stimulated migration and 12-HETE biosynthesis. Structurally diverse drugs at concentrations that inhibited HETE biosynthesis inhibited ESP-induced migration. The concentrations that decreased migration activity by one-half were 5 microM NDGA, 10 microM ETYA, and 150 microM BW755C. Aspirin and indomethacin at concentrations reported to inhibit prostaglandin biosynthesis did not substantially inhibit ESP activity, but concentrations of indomethacin above 20 microM caused concentration-dependent inhibition of migration. The selective lipoxygenases inhibitor 134,7,10,13-eicosatetraynoic acid was more potent than ETYA in inhibition of ESP-induced migration, and the selective cyclooxygenase inhibitor 6,9,12-octadecatriynoic acid did not effect inhibition. These results are consistent with the hypothesis that stimulation of eosinophils by the lymphokine ESP involves the generation of lipoxygenase products from arachidonic acid, which positively and negatively regulate the migratory activities of these cells.     

Growth stimulation by PGE2 and EGF activates cyclic AMP-dependent and -independent pathways in primary cultures of mouse mammary epithelial cells

Mammary epithelial cells from virgin Balb/c mice were isolated by collagenase digestion and cultured within collagen gels in serum-free basal medium containing insulin (10 micrograms/ml). Previous work has shown that linoleate or its metabolite, prostaglandin E2 (PGE2), stimulate the growth of these cells only in the presence of a growth stimulant such as epidermal growth factor (EGF). Since PGE2 can stimulate cyclic AMP (cAMP) production, the role of cAMP in linoleate and EGF-stimulated growth was examined. The cAMP phosphodiesterase inhibitor, IBMX (0.1 mM), was found to augment growth when cells were cultured in the presence of both EGF and linoleate or PGE2, but not either factor alone. These results indicated that EGF does not stimulate proliferation via cyclic AMP mediated events but could synergize with cAMP events if cAMP levels were elevated by PGE2. When assayed in cells plated on top of collagen-coated culture dishes, cellular cyclic AMP levels were stimulated by PGE2, but only marginally by EGF. Although the stimulation of endogenous cAMP by PGE2 and IBMX was insufficient to stimulate growth in the absence of EGF, exogenous dibutyryl-cAMP (greater than 100 micrograms/ml) was able to do so showing that a sustained, and high level of cAMP (greater than 100 micrograms/ml) could stimulate growth in insulin-containing basal medium. EGF was capable of enhancing the cellular sensitivity to dibutyryl-cAMP but the converse was not observed. cAMP stimulation of growth was dependent upon a superphysiological concentration of insulin (10 micrograms/ml) or a physiological concentration of somatomedin-C. These results indicate that the proliferation of mouse mammary epithelial cells can be stimulated separately or in synergism by cAMP-dependent or -independent events.