The in vitro inhibition of growth of intracellular Listeria monocytogenes by lymphocyte products

The listeria-inhibiting activity of culture supernatants from listeria-immune and nonimmune lymphocytes was assessed on listeria-infected macrophage cultures. It was found that supernatant from listeria-immune lymphocyte cultures stimulated in vitro with antigen was markedly inhibitory to the multiplication of intracellular listeria. Some inhibitory activity was also evident in supernatant from antigen-stimulated non-immune lymphocyte cultures. Supernatant from listeria-immune lymphocytes stimulated in vivo with antigen was capable of inhibiting listeria. Some inhibitory activity was still evident upon dilution of active supernatant at 1:100.     

Responses of the Smooth Muscle Membrane of Guinea Pig Jejunum Elicited by Field Stimulation

Field stimulation of the jejunum elicited successively an action potential of spike form, a slow excitatory depolarization, a slow inhibitory hyperpolarization, and a postinhibitory depolarization as a rebound excitation. The slow depolarization often triggered the spike. The inhibitory potential showed lower threshold than did the excitatory potential. Both the excitatory potentials were abolished by atropine and tetrodotoxin. Effective membrane resistance measured by the intracellular polarizing method was reduced during the peak of the excitatory potential, but the degree of reduction was smaller than that evoked by iontophoretic application of acetylcholine. Conditioning hyperpolarization of the muscle membrane modified the amplitude of the excitatory potential. The estimated reversal potential level for the excitatory potenialt was about 0 mv. No changes could be observed in the amplitude of the inhibitory potential when hyperpolarization was induced with intracellularly applied current. Low [K]_o and [Ca]_o blocked the generation of the excitatory potential but the amplitude of the inhibitory potential was enhanced in low [K]_o. Low [Ca]_o and high [Mg]_o had no effect on the inhibitory potential.     

Phosphoenolpyruvate synthesis by fetal guinea-pig liver mitochondria

Liver mitochondria isolated from fetal and newborn guinea pigs synthesized phosphoenolpyruvate at 4–6 nmol/min per mg protein with 2 mM malate, succinate, and α-ketoglutarate as substrates. These rates were 90–110% of that by adult liver mitochondria and were not substantially altered in the second half of gestation or within 24 h after birth. Both palmitoyl- and octanoylcarnitine were inhibitory to phosphoenolpyruvate synthesis in adult and fetal preparations, but free octanoate was inhibitory only in adult liver mitochondria.     

Purification and characterization of plantaricin Y,a novel bacteriocin produced by Lactobacillus plantarum 510

Lactobacillus plantarum 510, previously isolated from a koshu vineyard in Japan, was found to produce a bacteriocin-like inhibitory substance which was purified and characterized. Mass spectrometry analysis showed that the mass of this bacteriocin is 4,296.65 Da. A partial sequence, NH₂- SSSLLNTAWRKFG, was obtained by N-terminal amino acid sequence analysis. A BLAST search revealed that this is a unique sequence; this peptide is thus a novel bacteriocin produced by Lactobacillus plantarum 510 and was termed plantaricin Y. Plantaricin Y shows strong inhibitory activity against Listeria monocytogenes BCRC 14845, but no activity against other pathogens tested. Bacteriocin activity decreased slightly after autoclaving (121 °C for 15 min), but was completely inactivated by protease K. Furthermore, trypsin-digested bacteriocin product fragments retained activity against L. monocytogenes BCRC 14845 and exhibited a different inhibitory spectrum.     

Different course of proteolytic inhibitory activity and proteolytic activity in Galleria mellonella larvae infected by Nosema algerae and Vairimorpha heterosporum

The activity of protease inhibitors and proteases was studied in the hemolymph, gut, and fat body of 7th-instar larvae of Galleria mellonella infected by two microsporidia, Nosema algerae and Vairimorpha heterosporum. The increase in inhibitory activity in the hemolymph was substantial, and coincided with the development of the disease. The increase in inhibitory activity in the gut was almost doubled by N. algerae as compared with V. heterosporum, whereas the increase in inhibitory activity in fat body was found only in V. heterosporum-infected larvae. The course of proteolytic activity followed an inverse pattern to the elevated activity of inhibitors in the gut and the fat body, and rose only in moribund larvae at the end of the course of V. heterosporum infection. The differences in the pattern of proteases and inhibitors reflect the organ specificity of each of the microsporidia.     

Microrespirometric characterization of activated sludge inhibition by copper and zinc

We have developed a novel microrespirometric method to characterize the inhibitory effects due to heavy metals on activated sludge treatment. This method was based on pulse dynamic respirometry and involved the injection of several pulses of substrate and inhibitors, of increasing concentration. Furthermore, we evaluated the inhibitory effects of heavy metals (copper and zinc), substrate and biomass concentrations, and pH on activated sludge activity. While higher biomass concentrations counteracted the inhibitory effects of both copper and zinc, higher substrate concentrations predominantly augmented the inhibitory effect of copper but no significant change in inhibition by zinc was observed. pH had a clear but relatively small effect on inhibition, partially explained by thermodynamic speciation. We determined the key kinetic parameters; namely, the half saturation constant (K _S ) and the maximum oxygen uptake rate (OUR _max ). The results showed that higher heavy metal concentrations substantially decreased K _S and OUR _max suggesting that the inhibition was uncompetitive.     

Inhibition of Listeria monocytogenes by Food-Borne Yeasts

Many bacteria are known to inhibit food pathogens, such as Listeria monocytogenes, by secreting a variety of bactericidal and bacteriostatic substances. In sharp contrast, it is unknown whether yeast has an inhibitory potential for the growth of pathogenic bacteria in food. A total of 404 yeasts were screened for inhibitory activity against five Listeria monocytogenes strains. Three hundred and four of these yeasts were isolated from smear-ripened cheeses. Most of the yeasts were identified by Fourier transform infrared spectroscopy. Using an agar-membrane screening assay, a fraction of approximately 4% of the 304 red smear cheese isolates clearly inhibited growth of L. monocytogenes. Furthermore, 14 out of these 304 cheese yeasts were cocultivated with L. monocytogenes WSLC 1364 on solid medium to test the antilisterial activity of yeast in direct cell contact with Listeria. All yeasts inhibited L. monocytogenes to a low degree, which is most probably due to competition for nutrients. However, one Candida intermedia strain was able to reduce the listerial cell count by 4 log units. Another four yeasts, assigned to C. intermedia (three strains) and Kluyveromyces marxianus (one strain), repressed growth of L. monocytogenes by 3 log units. Inhibition of L. monocytogenes was clearly pronounced in the cocultivation assay, which simulates the conditions and contamination rates present on smear cheese surfaces. We found no evidence that the unknown inhibitory molecule is able to diffuse through soft agar.     

Inhibition of the growth of two blue-green algae species (Microsystis aruginosa and Anabaena spiroides) by acidification treatments using carbon dioxide

The effect of pH adjusted by aeration with carbon dioxide (CO₂) on the growth of two species of blue-green algae, Microcystis aeruginosa and Anabaena spiroides, was investigated. Three conditions (pH 5.5, 6.0 and 6.5) were found to have significant inhibitory effects on the growth of the two algae species when acidification treatment was conducted during the logarithmic phase. Differences in the inhibition effect of acidification existed between the two species algae. The tolerance of M. aeruginosa to these conditions was also investigated. The results indicated that M. aeruginosa was inhibited significantly, but not dead at pH 6.5, whereas death occurred at pH 5.5 and 6.0. The greatest inhibitory effect of acidification treatment conducted during the stable breeding phase of M. aeruginosa occurred at pH 5.5, while no inhibitory effect was found at pH 6.5.     

Reduction of leptin secretion by soy isoflavonoids in murine adipocytes in vitro

Daidzein and genistein are the main aglycones of soy isoflavonoid, and have many useful activities in vitro and in vivo. However, equol, a metabolite of daidzein in vivo, has attracted attention due to its stronger activity than that of the naturally occurring isoflavonoids. We subjected the soy isoflavonoids, including the naturally occurring (S)-equol, to mouse adipocytes, and compared the inhibitory activity on the leptin secretion. Equol, daidzein and genistein inhibited the leptin secretion, whereas O-desmethylangolensin had a lower activity. The inhibitory activity of the isoflavones was not affected by the addition of an iNOS inhibitor and an estrogen.     

Competitive inhibition of cellobiohydrolase I by manno-oligosaccharides

In the hydrolysis of softwood, significant amounts of manno-oligosaccharides (MOS) are released from mannan, the major hemicelluloses in softwood. However, the impact of MOS on the performance of cellulases is not yet clear. In this work, the effect of mannan and MOS in cellulose hydrolysis by cellulases, especially cellobiohydrolase I (CBHI) from Thermoascus aurantiacus (Ta Cel7A), was studied. The glucose yield of Avicel decreased with an increasing amount of added mannan. Commercial cellulases contained mannan hydrolysing enzymes, and β-glucosidase played an important role in mannan hydrolysis. Addition of 10 mg/ml mannan reduced the glucose yield of Avicel (at 20 g/l) from 40.1 to 24.3%. No inhibition of β-glucosidase by mannan was observed. The negative effects of mannan and MOS on the hydrolytic action of cellulases indicated that the inhibitory effect was at least partly attributed to the inhibition of Ta Cel7A (CBHI), but not on β-glucosidase. Kinetic experiments showed that MOS were competitive inhibitors of the CBHI from T. aurantiacus, and mannobiose had a stronger inhibitory effect on CBHI than mannotriose or mannotetraose. For efficient hydrolysis of softwood, it was necessary to add supplementary enzymes to hydrolyze both mannan and MOS to less inhibitory product, mannose.     

Inhibition of chymase activity by phosphoglycerides

The activity of chymase was markedly inhibited by phosphoglycerides such as phosphatidic acid, phosphatidylserine, and phosphatidylinositol, but was not affected by acylglycerides, phosphoglyceroserine, serine, inositol, or glycerol. These results suggest that both the nonpolar hydrophobic hydrocarbon tails and the polar hydrophilic head are essential for the inhibitory effects of phosphoglycerides. Binding of a primary amine to an anionic polar head of phosphatidic acid, such as in phosphatidylserine and phosphatidylethanolamine, slightly decreased the inhibitory effect of phosphatidic acid and, conversely, binding of a strong cation to the head, such as in phosphatidylcholine, resulted in its activation of chymase. Phosphatidic acid containing an unsaturated fatty acid, such as dioleoyl phosphatidic acid, caused the same extent of inhibition as natural phosphatidic acid from bovine brain, but was 20 times more inhibitory than phosphatidic acid containing a saturated fatty acid, such as distearoyl phosphatidic acid. The inhibition by phosphatidylserine was noncompetitive and pseudoirreversible, and the K_i value was 0.54 μm. The inhibition of chymase by phosphatidylserine was pH dependent, being strong at pH 8.5 to 9.5 but weak below pH 7.5. Phosphatidylserine specifically inhibited chymase and elastase; it did not inhibit the other chymotrypsin-type serine endopeptidases tested, trypsin, papain, collagenase, carboxypeptidase A, or cathepsin D.     

Co-fermentation of lignocellulose-based glucose and inhibitory compounds for lipid synthesis by Rhodococcus jostii RHA1

The recalcitrant nature of lignocellulosic biomass entails pretreatment during which multiple byproducts (e.g., weak acids, furan derivatives, lignin-derived compounds) are generated. Such byproducts are generally inhibitory to fuel-producing microorganisms. In this study, lignin-derived monomers and acetate were co-fermented with glucose by Rhodococcus jostii RHA1 for lipid synthesis. The ability of R. jostii RHA1 to utilize acetate and representative lignin-derived monomers, namely p-coumaric acid, ferulic acid, 4-hydroxylic acid, and vanillic acid, were tested. The experimental results showed that R. jostii RHA1 utilized individual lignin monomers in varying degrees. The mixtures of inhibitory compounds at different levels showed higher toxicity than individual compounds, indicating synergistic effects of these monomers. When the mixture contained lower levels of glucose (5 g/L or below), adaptive-evolved (AE) R. jostii RHA1 utilized such inhibitory mixtures better for lipid synthesis. When the glucose levels were increased to 20 g/L or above, adaption evolution appeared to shorten the lag phase of co-fermentation but not necessarily enhance lipid production. This study demonstrated that R. jostii RHA1 was capable of utilizing commonly unfavorable carbon sources for lipid synthesis, which would also serve as a means to in situ detoxify inhibitory compounds.     

Enhanced species-specific chemical control of harmful and non-harmful algal bloom species by the thiazolidinedione derivative TD49

Culture experiments involving 23 algae strains were conducted to evaluate the algicidal effects of a newly developed algicidal thiazolidinedione (TD) derivative (TD49) on non-harmful and harmful algal bloom (HAB) species. We also assessed the effect of various concentrations of TD49 on various growth phases (lag, logarithmic, and stationary) of the harmful algae Heterocapsa circularisquama (Dinophyceae) and Heterosigma akashiwo (Raphidophyceae; hereafter, Heterosigma) and the non-harmful diatoms Skeletonema costatum and Chaetoceros didymus. The inhibitory ratios (%) for H. circularisquama and Heterosigma at 2.0 μM TD49 were significantly higher than those at other concentrations, and the inhibitory ratio varied depending on growth phase and species as follows: logarithmic?≥?stationary?>?lag phase for H. circularisquama and logarithmic?≥?lag?>?stationary phase for Heterosigma. Although the inhibitory ratios for C. didymus were similar to those for the two harmful algae (H. circularisquama and Heterosigma), inhibitory effects on S. costatum were not apparent at >2.0 μM in any growth phase. The algicidal activity of TD49 on the harmful and non-harmful algae was as follows: unarmored HAB species?>?armored HAB species?>?diatom species?>?cryptophyte species. TD49 was algicidal to most HABs but had a little inhibitory effect on some non-harmful algae, implying that TD49 has selective algicidal activity. Our results indicate that TD49 is potentially of use in the control of HAB species within semi-enclosed bays.     

Plasmodium fragile: Inhibition of cultures by serum from Rhesus monkeys immunized with homologous parasites

Rhesus monkeys, Macaco mulatto, that had previously been immunized with the Nilgiri strain of Plasmodium fragile grown in culture, together with control monkeys with and without inoculation of Freund's adjuvant, were challenged with cultured parasites. After treatment with chloroquine, the monkeys were rechallenged. Serum specimens from three immunized monkeys caused a specific, dose-dependent inhibition of parasite growth in culture. Fifty percent inhibition of in vitro growth was obtained using 5% immune serum combined with 10% normal rhesus serum. The specific inhibitory component of immune serum was shown to be IgG antibody. Results of the study demonstrated that there is good correlation between the inhibitory activity of immune serum, parasite growth in vitro, the in vivo response to challenge, and the indirect fluorescent antibody titer.     

Control of Listeria monocytogenes in a Biofilm by Competitive-Exclusion Microorganisms

Biofilms from drains in food processing facilities with a recent history of no detectable Listeria monocytogenes in floor drains were cultured for microorganisms producing antilisterial metabolites. A total of 413 microbial isolates were obtained from 12 drain biofilm samples and were assayed at 15 and 37°C for activities that were bactericidal or inhibitory to L. monocytogenes, by two agar plate assays. Twenty-one of 257 bacterial isolates and 3 of 156 yeast isolates had antilisterial activity. All 24 isolates which produced metabolites inhibitory to L. monocytogenes were assayed for antilisterial activity in coinoculated broth cultures containing tryptic soy broth with yeast extract (TSB-YE). A five-strain mixture of 10³ CFU of L. monocytogenes/ml and 10⁵ CFU of the candidate competitive-exclusion microorganism/ml was combined in TSB-YE and incubated at 37°C for 24 h, 15°C for 14 days, 8°C for 21 days, and 4°C for 28 days. Substantial inhibition of L. monocytogenes growth (4 to 5 log CFU/ml) was observed for nine bacterial isolates at 37°C, two at 15 and 8°C, and three at 4°C. The inhibitory isolates were identified as Enterococcus durans (six isolates), Lactococcus lactis subsp. lactis (two isolates), and Lactobacillus plantarum (one isolate). The anti-L. monocytogenes activity of these isolates was evaluated in biofilms of L. monocytogenes on stainless steel coupons at 37, 15, 8, and 4°C. Results revealed that two isolates (E. durans strain 152 and L. lactis subsp. lactis strain C-1-92) were highly inhibitory to L. monocytogenes (growth inhibition of >5 log₁₀ CFU of L. monocytogenes/cm²). These two bacterial isolates appear to be excellent competitive-exclusion candidates to control L. monocytogenes in biofilms at environmental temperatures of 4 to 37°C.     

Inhibition of Aspergillus fumigatus and Its Biofilm by Pseudomonas aeruginosa Is Dependent on the Source,Phenotype and Growth Conditions of the Bacterium

Aspergillus fumigatus (Af) and Pseudomonas aeruginosa (Pa) are leading fungal and bacterial pathogens, respectively, in many clinical situations. Relevant to this, their interface and co-existence has been studied. In some experiments in vitro, Pa products have been defined that are inhibitory to Af. In some clinical situations, both can be biofilm producers, and biofilm could alter their physiology and affect their interaction. That may be most relevant to airways in cystic fibrosis (CF), where both are often prominent residents.We have studied clinical Pa isolates from several sources for their effects on Af, including testing involving their biofilms. We show that the described inhibition of Af is related to the source and phenotype of the Pa isolate. Pa cells inhibited the growth and formation of Af biofilm from conidia, with CF isolates more inhibitory than non-CF isolates, and non-mucoid CF isolates most inhibitory. Inhibition did not require live Pa contact, as culture filtrates were also inhibitory, and again non-mucoid>mucoid CF>non-CF. Preformed Af biofilm was more resistant to Pa, and inhibition that occurred could be reproduced with filtrates. Inhibition of Af biofilm appears also dependent on bacterial growth conditions; filtrates from Pa grown as biofilm were more inhibitory than from Pa grown planktonically. The differences in Pa shown from these different sources are consistent with the extensive evolutionary Pa changes that have been described in association with chronic residence in CF airways, and may reflect adaptive changes to life in a polymicrobial environment.     

Inhibition of compensatory renal growth by the N-terminus of a sheep-derived peptide

The N-terminal sequence of a novel sheep-derived peptide with growth inhibitory activity has been obtained. The N-terminal fragment was chemically synthesised and designated EPL001. The kidney was chosen as the first mammalian system in which to study EPL001 since kidney growth can be accurately quantified following a surgical reduction in renal mass. Cell proliferation was measured in mouse collecting duct kidney (MCDK) cells stimulated with insulin-like growth factor I (IGF-I). Compensatory renal growth (CRG) was induced in Wistar rats and either EPL001 or an EPL001 antibody delivered by continuous renal tissue infusion. Mouse monoclonal antibodies to EPL001 were generated for immunoneutralisation, rabbit polyclonal antibodies were generated for immunohistochemistry. EPL001 had no apparent effect on IGF-I stimulated cell proliferation in MCDK cells in vitro, yet provoked a dose-dependent inhibition of CRG in vivo. An EPL001 antibody potentiated CRG, in the absence of exogenous EPL001, consistent with an inhibitory role in kidney growth for an endogenous peptide containing the EPL001 sequence. Tubular staining for epitopes to the EPL001 sequence was detected in normal human kidney sections and enhanced in renal cell carcinoma. Results support the presence of growth inhibitory activity in the N-terminus of a sheep-derived peptide with evidence for both its presence and endogenous activity in the kidney. Attempts to further characterise its structure and activity are ongoing.     

Isolation of citroylformic acid-gamma-lactone from a commerical batch of oxaloacetic acid: inhibition of apotyrosine aminotransferase conversion to holoenzyme by this substance.

Citroylformic acid-γ-lactone (CFA, 1-keto-2,4-dihydroxy-4-carboxyadipenoic acid(2–3)-1,4-lactone), isolated from a commercial batch of oxaloacetate, inhibited conversion of rat liver apotyrosine aminotransferase (EC 2.6.1.5) to holoenzyme. Using partially purified enzyme, the K_i was determined to be less than 0.7 mm. A more definitive K_i was difficult to obtain because at pH 7 CFA had a half-life of about 2 hr. Inhibition of the enzyme by CFA was stereospecific and reversible; the S (?) stereoisomer was approximately 10 times more inhibitory than its R(+) antipode, and over 90% of inhibited enzyme was recoverable after overnight dialysis. Preineubation of apotyrosine aminotransferase with its coenzyme (pyridoxal phosphate) prevented inhibition by CFA, and a substantial fraction of enzyme that had been inhibited by CFA could be readily reactivated by addition of high concentrations of pyridoxal phosphate. Studies with inhibitor analogs indicated that both a partially unsaturated lactone ring and a stereospecific carboxymethyl group are required for maximal inhibitory activity. The sodium salts of citroylformic acid and oxalopyruvic acid, formed by the hydrolysis of their respective lactones, were not inhibitory; 1-keto-2,4-dihydroxy-4-carboxyadipic acid-γ-lactone and little inhibitory activity, and 1-keto-2,4-dihydroxyglutarenoic acid-γ-lactone and 1-keto-2,4-dihydroxybutene-γ-lactone were somewhat better inhibitors than the R(+) stereoisomer of CFA. The possibility that CFA is a naturally occurring biological substance is discussed.     

Inhibition of glucuronokinase by substrate analogs

Glucuronokinase from Lilium longiflorum pollen was purified 30- to 40- fold on a blue dextran-Sepharose column. Substrate analogs were tested for inhibitory effects, and nucleotide substrate specificity of the enzyme was determined. Nine nucleotides were tested, and all were inhibitory when the substrate was ATP. ADP was competitive with ATP and had a K_i value of 0.23 mm. None of the other nucleotide triphosphates could effectively substitute for ATP as a nucleotide substrate. Ten mm dATP and ITP reacted only 3% as rapidly as 10 mm ATP, while the rates for 10 mm GTP, CTP, UTP, and TTP were less than 1%. The glucuronic acid analogs, methyl α-glucuronoside, methyl β-glucuronoside, β-glucuronic acid-1-phosphate, and 4-O-methylglucuronic acid were tested as possible enzyme inhibitors. The three methyl derivatives showed little or no inhibition. The β-glucuronic acid-1-phosphate was inhibitory, with 50% inhibition obtained at 1 to 3 mm depending on the concentration of the glucuronic acid. It is concluded that the glucuronic acid-binding site on the enzyme is highly selective.     

Methylprednisolone and isoproterenol inhibit airway smooth muscle proliferation by separate and additive mechanisms

To determine whether glucocorticoids and β-adrenoceptor agonists act independently to inhibit airway smooth muscle (ASM) proliferation, the present study investigated the effects of methylprednisolone (MP) and isoproterenol (ISO) alone and in combination on leukotriene D₄-induced ASM proliferation. MP and ISO had no effect on unstimulated ASM cell growth. In contrast, MP and ISO demonstrated dose-dependent inhibition of LTD₄-induced proliferation, and the inhibitory effect was additive for combinations of MP and ISO. The competitive cAMP receptor antagonist, Rp-cAMPS, ablated the ISO-induced inhibition but had no affect on the inhibitory response to MP. In cells exposed to both ISO and MP, Rp-cAMPS attenuated the growth inhibition to levels achieved by MP alone. Accordingly, these findings demonstrate that glucocorticoids and β-adrenergic agonists inhibit LTD₄-induced ASM proliferation, and that their inhibitory effects are mediated by different signaling pathways.