scholarly journals Characterization of Reutericyclin Produced by Lactobacillus reuteri LTH2584

2000 ◽  
Vol 66 (10) ◽  
pp. 4325-4333 ◽  
Author(s):  
Michael G. Gänzle ◽  
Alexandra Höltzel ◽  
Jens Walter ◽  
Günther Jung ◽  
Walter P. Hammes
Keyword(s):  
Staphylococcus Aureus ◽  
Antimicrobial Activity ◽  
Culture Supernatant ◽  
Lactobacillus Reuteri ◽  
Gel Filtration ◽  
Reversed Phase ◽  
Exchange Chromatography ◽  
Growth Media ◽  
Dependent Manner ◽  
Cell Extracts

ABSTRACT Lactobacillus reuteri LTH2584 exhibits antimicrobial activity that can be attributed neither to bacteriocins nor to the production of reuterin or organic acids. We have purified the active compound, named reutericyclin, to homogeneity and characterized its antimicrobial activity. Reutericyclin exhibited a broad inhibitory spectrum including Lactobacillus spp., Bacillus subtilis, B. cereus, Enterococcus faecalis, Staphylococcus aureus, and Listeria innocua. It did not affect the growth of gram-negative bacteria; however, the growth of lipopolysaccharide mutant strains ofEscherichia coli was inhibited. Reutericyclin exhibited a bactericidal mode of action against Lactobacillus sanfranciscensis, Staphylococcus aureus, and B. subtilis and triggered the lysis of cells of L. sanfranciscensis in a dose-dependent manner. Germination of spores of B. subtilis was inhibited, but the spores remained unaffected under conditions that do not permit germination. The fatty acid supply of the growth media had a strong effect on reutericyclin production and its distribution between producer cells and the culture supernatant. Reutericyclin was purified from cell extracts and culture supernatant of L. reuteri LTH2584 cultures grown in mMRS by solvent extraction, gel filtration, RP-C8 chromatography, and anion-exchange chromatography, followed by rechromatography by reversed-phase high-pressure liquid chromatography. Reutericyclin was characterized as a negatively charged, highly hydrophobic molecule with a molecular mass of 349 Da. Structural characterization (A. Höltzel, M. G. Gänzle, G. J. Nicholson, W. P. Hammes, and G. Jung, Angew. Chem. Int. Ed. 39:2766–2768, 2000) revealed that reutericyclin is a novel tetramic acid derivative. The inhibitory activity of culture supernatant of L. reuteri LTH2584 corresponded to that of purified as well as synthetic reutericyclin.

scholarly journals Protein A-Mediated Multicellular Behavior in Staphylococcus aureus

2008 ◽  
Vol 191 (3) ◽  
pp. 832-843 ◽  
Author(s):  
Nekane Merino ◽  
Alejandro Toledo-Arana ◽  
Marta Vergara-Irigaray ◽  
Jaione Valle ◽  
Cristina Solano ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Biofilm Formation ◽  
Protein A ◽  
Surface Proteins ◽  
Growth Media ◽  
Dependent Manner ◽  
Chronic Infections ◽  
The Matrix ◽  
Implanted Medical Devices ◽  
Biofilm Development

ABSTRACT The capacity of Staphylococcus aureus to form biofilms on host tissues and implanted medical devices is one of the major virulence traits underlying persistent and chronic infections. The matrix in which S. aureus cells are encased in a biofilm often consists of the polysaccharide intercellular adhesin (PIA) or poly-N-acetyl glucosamine (PNAG). However, surface proteins capable of promoting biofilm development in the absence of PIA/PNAG exopolysaccharide have been described. Here, we used two-dimensional nano-liquid chromatography and mass spectrometry to investigate the composition of a proteinaceous biofilm matrix and identified protein A (spa) as an essential component of the biofilm; protein A induced bacterial aggregation in liquid medium and biofilm formation under standing and flow conditions. Exogenous addition of synthetic protein A or supernatants containing secreted protein A to growth media induced biofilm development, indicating that protein A can promote biofilm development without being covalently anchored to the cell wall. Protein A-mediated biofilm formation was completely inhibited in a dose-dependent manner by addition of serum, purified immunoglobulin G, or anti-protein A-specific antibodies. A murine model of subcutaneous catheter infection unveiled a significant role for protein A in the development of biofilm-associated infections, as the amount of protein A-deficient bacteria recovered from the catheter was significantly lower than that of wild-type bacteria when both strains were used to coinfect the implanted medical device. Our results suggest a novel role for protein A complementary to its known capacity to interact with multiple immunologically important eukaryotic receptors.

Staphylococcus aureus stimulates neutrophil recruitment by stimulating interleukin-8 production in dog trachea

1995 ◽  
Vol 268 (1) ◽  
pp. L85-L94 ◽  
Author(s):  
P. P. Massion ◽  
C. A. Hebert ◽  
S. Leong ◽  
B. Chan ◽  
H. Inoue ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Culture Supernatant ◽  
Chemotactic Activity ◽  
Neutrophil Recruitment ◽  
Interleukin 8 ◽  
Biologically Active ◽  
Time Dependent ◽  
Dependent Manner ◽  
Tracheal Epithelial Cells ◽  
Tracheal Epithelial

The present study examined whether neutrophil recruitment in dog airways by Staphylococcus aureus is mediated by interleukin-8 (IL-8). S. aureus culture supernatant was superfused into an isolated tracheal segment in six dogs, and neutrophil recruitment and IL-8 concentrations were measured in the superfusate. Dog IL-8 was cloned, expressed in Escherichia coli, purified by chromatography, and shown to be biologically active. With the use of an enzyme-linked immunoabsorbent assay (ELISA) for the measurement of dog IL-8, we showed that S. aureus supernatant induced neutrophil recruitment and increased IL-8 concentration in the superfusate in a time-dependent manner. The chemotactic activity present in the superfusate 6 h after superfusion with S. aureus was inhibited by an anti-IL-8 antibody. S. aureus supernatant also stimulated IL-8 production and gene expression by cultured canine tracheal epithelial cells. These results provide evidence that IL-8 plays a major role in S. aureus-induced neutrophil recruitment in the airways by stimulating IL-8 production in airway cells

scholarly journals β-Carotene-15,15′-dioxygenase (EC 1.13.11.21) isolation reaction mechanism and an improved assay procedure

1994 ◽  
Vol 72 (3) ◽  
pp. 397-414 ◽  
Author(s):  
Jannine Devery ◽  
B. V. Milborrow
Keyword(s):  
Guinea Pig ◽  
Intestinal Mucosa ◽  
Specific Activity ◽  
Gel Filtration ◽  
Tween 80 ◽  
Reversed Phase ◽  
Exchange Chromatography ◽  
Protein Aggregate ◽  
Β Carotene ◽  
Dioxygenase Activity

βCarotene-15,15′-dioxygenase (EC 1.13.11.21; β-carotene dioxygenase) activity in extracts from guinea-pig intestinal mucosa was assayed by supplying [15,15′-14C2l- or [15,15′-3H2]β-carotene dissolved in Tween 80. Methods were developed to minimize the breakdown of labelled β-carotene and β-carotene cleavage products during the isolation procedure. Antioxidants and unlabelled carriers were added to extracting solvents and C18 Sep-Pak cartridges were used to isolate the remaining β-carotene and retinaldehyde, which was the only cleavage product detected. The labelled material produced by the enzyme was analysed by either normal-phase TLC or reversed-phase HPLC and characterized chemically as retinaldehyde. The lack of other labelled up-carotenals isolated in these experiments and the formation of between 1.5 and 2 mol retinaldehyde/mol β-carotene consumed confirm the central cleavage mechanism for the enzyme's action. More β-carotene dioxygenase activity was obtained from guinea-pig mucosa than from chicken or pig intestinal mucosa. The β-carotene dioxygenase was obtained as a soluble enzyme which was partially purified by gel filtration and ion-exchange chromatography to a specific activity of 0.6 nmol retinaldehyde formedlmg protein per h. The formation of a lipid-protein aggregate containing the β-carotene dioxygenase activity, which has been reported to be present in the exclusion volume of Sephadex columns, was avoided if the mucosal serapings were homogenized in buffer at a proportion of 1:4 (w/v).

scholarly journals Characterization of β-galactosidase and α-galactosidase activities from the halophilic bacterium Gracilibacillus dipsosauri

2021 ◽  
Vol 71 (1) ◽  
Author(s):  
Charles E. Deutch ◽  
Amy M. Farden ◽  
Emily S. DiCesare
Keyword(s):  
Gel Filtration ◽  
Halophilic Bacterium ◽  
Peptide Sequencing ◽  
Exchange Chromatography ◽  
Galactosidase Activity ◽  
Ph Optimum ◽  
Whole Cells ◽  
Cell Extracts ◽  
Genes Encoding ◽  
Hydrolysis Of

Abstract Purpose Gracilibacillus dipsosauri strain DD1 is a salt-tolerant Gram-positive bacterium that can hydrolyze the synthetic substrates o-nitrophenyl-β-d-galactopyranoside (β-ONP-galactose) and p-nitrophenyl-α-d-galactopyranoside (α-PNP-galactose). The goals of this project were to characterize the enzymes responsible for these activities and to identify the genes encoding them. Methods G. dipsosauri strain DD1 was grown in tryptic soy broth containing various carbohydrates at 37 °C with aeration. Enzyme activities in cell extracts and whole cells were measured colorimetrically by hydrolysis of synthetic substrates containing nitrophenyl moieties. Two enzymes with β-galactosidase activity and one with α-galactosidase activity were partially purified by ammonium sulfate fractionation, ion-exchange chromatography, and gel-filtration chromatography from G. dipsosauri. Coomassie Blue-stained bands corresponding to each activity were excised from nondenaturing polyacrylamide gels and subjected to peptide sequencing after trypsin digestion and HPLC/MS analysis. Result Formation of β-galactosidase and α-galactosidase activities was repressed by d-glucose and not induced by lactose or d-melibiose. β-Galactosidase I had hydrolytic and transgalactosylation activity with lactose as the substrate but β-galactosidase II showed no activity towards lactose. The α-galactosidase had hydrolytic and transgalactosylation activity with d-melibiose but not with d-raffinose. β-Galactosidase I had a lower Km with β-ONP-galactose as the substrate (0.693 mmol l−1) than β-galactosidase II (1.662 mmol l−1), was active at more alkaline pH, and was inhibited by the product d-galactose. β-Galactosidase II was active at more acidic pH, was partially inhibited by ammonium salts, and showed higher activity with α-PNP-arabinose as a substrate. The α-galactosidase had a low Km with α-PNP-galactose as the substrate (0.338 mmol l−1), a pH optimum of about 7, and was inhibited by chloride-containing salts. β-Galactosidase I activity was found to be due to the protein A0A317L6F0 (encoded by gene DLJ74_04930), β-galactosidase II activity to the protein A0A317KZG3 (encoded by gene DLJ74_12640), and the α-galactosidase activity to the protein A0A317KU47 (encoded by gene DLJ74_17745). Conclusions G. dipsosauri forms three intracellular enzymes with different physiological properties which are responsible for the hydrolysis of β-ONP-galactose and α-PNP-galactose. BLAST analysis indicated that similar β-galactosidases may be formed by G. ureilyticus, G. orientalis, and G. kekensis and similar α-galactosidases by these bacteria and G. halophilus.

scholarly journals Karakterisasi Plantarisin IIA-1A5 sebagai Antimikroba dan Evaluasi Aktivitas Sediaan Kering Beku Terenkapsulasi

2020 ◽  
Vol 9 (1) ◽  
pp. 30
Author(s):  
Mochammad Sriduresta Soenarno ◽  
Irma Isnafia Arief ◽  
Cece Sumantri ◽  
Epi Taufik ◽  
Lilis Nuraida
Keyword(s):  
Molecular Weight ◽  
Staphylococcus Aureus ◽  
Antimicrobial Activity ◽  
Lactobacillus Plantarum ◽  
Exchange Chromatography ◽  
Single Band ◽  
Food Preservatives ◽  
Maldi Tof ◽  
Freeze Dried ◽  
Sds Page

Bakteriosin adalah peptida dengan aktivitas antibakteri yang diproduksi oleh bakteri asam laktat dan digunakan sebagai pengawet alami. Penelitian sebelumnya menunjukkan bahwa Lactobacillus plantarum IIA-1A5 memproduksi bakteriosin yang diberi nama Plantarisin IIA-1A5 pada medium pertumbuhan yang dibuat dari whey yang diperkaya skim. Untuk aplikasi sebagai pengawet alami dan untuk memperbaiki masa simpan dan aktivitas anti mikrobanya, plantarisin perlu dienkapsulasi dan dikeringbekukan. Tujuan dari penelitian ini adalah untuk mengkarakterisasi dan mengevaluasi aktivitas antimikroba dari sediaan plantarisin IIA-1A5 yang terpurifikasi parsial dan terenkapsulasi kering beku. Ekstraksi dan purifikasi dari bakteriosin dimulai dengan presipitasi dengan ammonium sulfat, yang diikuti dengan dialysis, dan penukar kation kromatografi. Purifikasi parsial dari plantarisin kemudian dimikroenkapsulasi dengan maltodextrin kemudian dilanjutkan dengan proses kering beku. Berdasarkan pada SDS-PAGE, fraksi protein ke-7 (F7) dari plantarisin yang dipurifikasi parsial memiliki pita tunggal dan berat molekul sekitar 9,65 kDa. Konfirmasi lebih lanjut dengan menggunakan MALDI-TOF MS, ternyata pita tunggal tersebut terdiri dari 5 peptida yang diidentifikasi berbobot molekul masing-masing sebagai berikut 5,5, 7,80, 7,96, 9,09, dan 9,27 kDa. Plantarisin kering beku memiliki aktivitas antimikroba terhadap Staphylococcus  aureus tiga kali lipat dibandingkan dengan aktivitas antimikroba dari supernatan bebas sel, dan lebih tinggi dibandingkan dengan nisin, namun kurang bila dibandingkan dengan antibiotik ampisilin dan penisilin. Kesimpulannya, aktivitas antimikroba plantarisin kering beku dapat ditentukan dan lebih tinggi dibandingkan dengan nisin, ampisilin dan penisilin.Characterization of Plantarisin IIA-1A5 as Antimicrobial subtances and Evaluation of Acitivity of Freeze-dried Microencapsulated PreparationAbstractBacteriocins are peptides with antibacterial activity produced by lactic acid bacteria and used as natural preservatives. Previous studies showed that Lactobacillus plantarum IIA-1A5 produces bacteriocin named plantaricin IIA-1A5 in the medium consisting whey enriched with skim milk. For application as food preservatives and to improve its shelf-lie and activity, plantaricin was needed to be microencapsulated and freeze dried. The objective of this research was to characterize and evaluate the activity of partially purified freeze dried microencapsulated plantaricin IIA-1A5. Characterisation of partially purified plantaricin IIA-IA5 includes the identification of active fractions and molecular weight, evaluation of activity at different stage of purification and evaluation of antimicrobial activity of freeze dried microencapsulated plantaricin IIA-IA5. Extraction and prificafication of the bacteriocins started with precipitacion with ammonium sulfate, followed by dialysis, and cation exchange chromatography. The partial purified of plantaricin was then microencapsulated in maltodextrin followed by freeze drying. Based on SDS-PAGE, the protein fraction F7 of partially purified plantaricin had a single band and molecular weight about 9.65 kDa. Further analyses using MALDI-TOF, it revealed that five peptides were identified from one single band plantaricin with molecular weight 5.5, 7.80, 7.96, 9.09, and 9.27 kDa, respectively. The freeze dried plantaricin freeze showed antimicrobial activity against Staphylococcus aureus three times stringer as compared to the activity of cell free supernatant, and was higher than nicin, but less than antibiotic ampicilin and penicilin. As concusion, the activity of freeze dried plantaricin could be determined and had a higher value than nicin, ampicilin and penicilin.

Inhibition of Urokinase by Complex Formation with Human Antithrombin III in Absence and Presence of Heparin

1978 ◽  
Vol 39 (03) ◽  
pp. 616-563 ◽  
Author(s):  
Inge Clemmensen
Keyword(s):  
Affinity Chromatography ◽  
Gel Electrophoresis ◽  
Antithrombin Iii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Hydrolytic Activity ◽  
Exchange Chromatography ◽  
Dependent Manner ◽  
Crossed Immunoelectrophoresis

SummaryHuman antithrombin III was purified from fresh human plasma by affinity chromatography on heparin-Sepharose®, affinity chromatography on concanavalin A Sepharose®, gel filtration on Ultrogel® AcA 34, ion exchange chromatography on DEAE A-50 Sephadex® and preparative agarose gel electrophoresis. The hydrolytic activity of urokinase (plasminogen activator from urine) on acetyl-glycyl-L-lysine methyl ester acetate (Ac-gly-lys-OMe Ac) was inhibited by antithrombin III in a slow time-dependent manner. Heparin accelerated the reaction between activator and inhibitor. Inhibition of catalytic activity was associated with the formation of an 1:1 molar complex between activator and inhibitor as revealed by sodium dodecyl sulphate polyacrylamide gel electrophoresis. The complex was also demonstrated by crossed Immunoelectrophoresis against anti-antithrombin III.

Isolation and partial characterization of boar proacrosin

1990 ◽  
Vol 55 (3) ◽  
pp. 846-853 ◽  
Author(s):  
Věra Jonáková ◽  
Brigita Vidimská ◽  
Jana Urbanová ◽  
Manfred Pavlík
Keyword(s):  
Amino Acid ◽  
High Performance ◽  
Gel Filtration ◽  
Reversed Phase ◽  
Exchange Chromatography ◽  
Relative Molecular Mass ◽  
Reducing Conditions ◽  
Apparent Homogeneity ◽  
Step Procedure

Boar proacrosin was purified to apparent homogeneity by a three-step procedure: gel filtration on Sephadex G-50 medium, ion-exchange chromatography on CM-Cellulose 32, and reversed-phase high-performance liquid chromatography on a C4 column. The relative molecular mass (Mr) of the proacrosin estimated by gel filtration was about 70 000, whereas the results of an electrophoretic experiment on SDS-polyacrylamide gel with copolymerized casein under non-reducing conditions indicated an Mr of 55 000-60 000. The proacrosin reproducibly migrated on the gel as a double band. When purified, it remained stable at pH 8.0 for 30 min. The amino-acid composition of the homogeneous proacrosin was determined, the N-terminal amino-acid sequence being Arg-Asp-X-Ala-Thr-X-X-Gly-Pro-X-Gly-.

scholarly journals Biosynthesis of glycosaminoglycans by cultured mastocytoma cells

1973 ◽  
Vol 134 (2) ◽  
pp. 455-463 ◽  
Author(s):  
Richard G. Lewis ◽  
Alice F. Spencer ◽  
Jeremiah E. Silbert
Keyword(s):  
Mast Cells ◽  
Chondroitin Sulphate ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Growth Media ◽  
Mastocytoma Cells ◽  
The Media ◽  
Sulphated Glycosaminoglycan ◽  
Testicular Hyaluronidase

Biosynthesis of glycosaminoglycans by several lines of cultured neoplastic mouse mast cells was studied by incorporation of [35S]sulphate (and in some cases [6-3H]glucosamine) into macromolecular materials found in both the cells and their growth media. Such intracellular and extracellular radioactively labelled materials (shown to be glycosaminoglycans by susceptibility to digestion with heparinase) were further characterized by ion-exchange chromatography and by digestion with testicular hyaluronidase and chondroitinase. All but one cell line produced chondroitin sulphate as the major sulphated glycosaminoglycan; the remainder of the glycosaminoglycan was heparin-like material. No [3H]hyaluronic acid was synthesized. Cells of a newly derived line, termed P815S, synthesized more glycosaminoglycan than the other lines. This glycosaminoglycan, found in both cells and growth medium, was almost entirely chondroitin 4-sulphate. No chondroitin 6-sulphate was found. The chondroitin 4-sulphate from the cells was shown by gel filtration to be smaller than the chondroitin 4-sulphate in the media of these cultures. This discovery of relatively high proportions of chondroitin 4-sulphate in these mastocytoma-derived cells is noteworthy, since mast cells have generally been considered to produce heparin as their major glycosaminoglycan.

scholarly journals 2594. Biofilm-Dispersed Staphylococcus aureus Exhibits a Distinct agr-Independent Host Interaction

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S901-S902
Author(s):  
Spencer Chang ◽  
Vance G Fowler ◽  
Batu K Sharma-Kuinkel ◽  
Felix Medie ◽  
Larry Park ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Inflammatory Responses ◽  
Glucose Deprivation ◽  
Reversed Phase ◽  
Host Immune System ◽  
Dependent Manner ◽  
Planktonic Cells ◽  
Independent Manner ◽  
Murine Bone Marrow ◽  
Dispersed Cells

Abstract Background Staphylococcus aureus biofilms are a common cause of persistent, life-threatening infections. Dispersal of S. aureus cells from established biofilm-based infections is crucial for dissemination within the host, but is poorly understood. We tested the hypothesis that biofilm dispersed S. aureus cells have distinct physiology from planktonic cells and are better equipped to evade host immunity in an agr-dependent manner. Methods Primary murine bone marrow-derived macrophages (BMDMs) were infected with planktonic and biofilm dispersed cells from S. aureus USA300 LAC wild type (WT) and USA300 LAC-agr knockout (KO). Biofilm dispersed cells were collected via glucose deprivation. Gentamicin protection assays were used to enumerate phagocytosed bacteria and fluorescence microscopy to quantify macrophage viability. A 26-plex immunoassay was used to screen for cytokines and chemokines. Reversed phase high-performance liquid chromatography was used to measure relative phenol-soluble modulin (PSM) levels from macrophage co-cultures. Results Compared with planktonic cells, biofilm-dispersed cells in both S. aureus WT and KO backgrounds exhibited: (1) ~10-fold less phagocytosis by BMDMs (p = 0.0003; Figure 1); (2) increased macrophage killing (23% vs. 8%; p = 0.0038; Figure 2); (3) stronger pro- (e.g., IFN-y, IL-2, IL-6, IL-17; Figure 3A) and anti- (e.g., IL-10, IL-4, IL-22; Figure 3B) inflammatory cytokine responses from macrophages (P < 0.05 for all); (4) significantly higher δ toxin PSM production (P = 0.0090; Figure 4) in WT background only. Conclusion S. aureus biofilm dispersed cells are physiologically distinct from planktonic cells and have a unique interaction with the host immune system. Dispersed cells are more resistant to phagocytosis, have a greater propensity to kill macrophages, and mount stronger pro- and anti-inflammatory responses in an agr-independent manner. Dispersed cells also have the ability to produce more δ toxin PSM via well-known agr-dependent pathways. Disclosures All authors: No reported disclosures.

Synthesis of modified human C-peptide and its fragments

1988 ◽  
Vol 53 (11) ◽  
pp. 2637-2644 ◽  
Author(s):  
Běla Bendlová ◽  
Michal Lebl ◽  
Pavel Štolba ◽  
Luboslav Stárka
Keyword(s):  
Solid Phase ◽  
Gel Filtration ◽  
Reversed Phase ◽  
Exchange Chromatography ◽  
Phase Method ◽  
Reversed Phase Hplc ◽  
Radioactive Label ◽  
C Peptide ◽  
Solid Phase Method ◽  
Internal Marker

Syntheses of the modified human C-peptide containing residues suitable for the introduction of the radioactive label (tyrosine) and internal marker for monitoring binding to carrier (norvaline) and five of its fragments are described. The syntheses were performed by solid phase method using either 9-fluorenylmethoxycarbonyl or tert-butyloxycarbonyl protecting groups. The products were purified by gel filtration, ion exchange chromatography and reversed phase HPLC. The reactivity of prepared peptides with antisera was determined and the modified C-peptide was found fully reactive.

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