Paenibacillus alvei MP1 as a Producer of the Proteinaceous Compound with Activity against Important Human Pathogens, Including Staphylococcus aureus and Listeria monocytogenes

An emerging need for new classes of antibiotics is, on the one hand, evident as antimicrobial resistance continues to rise. On the other hand, the awareness of the pros and cons of chemically synthesized compounds’ extensive use leads to a search for new metabolites in already known reservoirs. Previous research showed that Paenibacillus strain (P. alvei MP1) recovered from a buckwheat honey sample presented a wide spectrum of antimicrobial activity against both Gram-positive and Gram-negative pathogens. Recent investigation has confirmed that P. alvei MP1 (deposited at DDBJ/ENA/GenBank under the accession WSQB00000000) produces a proteinaceous, heat-stable compound(s) with the maximum antimicrobial production obtained after 18 h of P. alvei MP1 growth in LB medium at 37 °C with continuous shaking at 200 RPM. The highest activity was found in the 40% ammonium sulfate precipitate, with high activity also remaining in the 50% and 60% ammonium sulfate precipitates. Moderate to high antimicrobial activity that is insensitive to proteases or heat treatment, was confirmed against pathogenic bacteria that included L. monocytogenes FSL – X1-0001 (strain 10403S), S. aureus L1 – 0030 and E. coli O157: H7. Further studies, including de novo sequencing of peptides by mass spectrometry, are in progress.

Isolation of a Lactobacillus salivarius Strain and Purification of Its Bacteriocin, Which Is Inhibitory to Campylobacter jejuni in the Chicken Gastrointestinal System

ABSTRACT We evaluated anti-Campylobacter jejuni activity among >1,200 isolates of different lactic acid bacteria. Lactobacillus salivarius strain NRRL B-30514 was selected for further study. The cell-free, ammonium sulfate precipitate from the broth culture was termed the crude antimicrobial preparation. Ten microliters of the crude preparation created a zone of C. jejuni growth inhibition, and growth within the zone resumed when the crude preparation was preincubated with proteolytic enzymes. Bacteriocin OR-7, derived from this crude preparation, was further purified using ion-exchange and hydrophobic-interaction chromatography. The determined amino acid sequence was consistent with class IIa bacteriocins. Interestingly, OR-7 had sequence similarity, even in the C-terminal region, to acidocin A, which was previously identified from L. acidophilus and had activity only to gram-positive bacteria, whereas OR-7 had activity to a gram-negative bacterium. Bacteriocin activity was stable following exposure to 90°C for 15 min, also consistent with these types of antibacterial peptides. The purified protein was encapsulated in polyvinylpyrrolidone and added to chicken feed. Ten day-of-hatch chicks were placed in each of nine isolation units; two groups of birds were challenged with each of four C. jejuni isolates (one isolate per unit). At 7 days of age, one group of birds was treated with bacteriocin-emended feed for 3 days, and one group was left untreated. At 10 days of age, the birds were sacrificed and the challenge strain was enumerated from the bird cecal content. Bacteriocin treatment consistently reduced colonization at least one millionfold compared with levels found in the untreated groups. Nonchallenged birds were never colonized by C. jejuni. Bacteriocin from L. salivarius NRRL B-30514 appears potentially very useful to reduce C. jejuni in poultry prior to processing.

Reduction of Olive Knot Disease by a Bacteriocin from Pseudomonas syringae pv. ciccaronei

ABSTRACT A bacteriocin produced by Pseudomonas syringae pv. ciccaronei, used at different purification levels and concentrations in culture and in planta, inhibited the multiplication of P. syringae subsp. savastanoi, the causal agent of olive knot disease, and affected the epiphytic survival of the pathogen on the leaves and twigs of treated olive plants. Treatments with bacteriocin from P. syringae pv. ciccaronei inhibited the formation of overgrowths on olive plants caused by P. syringae subsp. savastanoi strains PVBa229 and PVBa304 inoculated on V-shaped slits and on leaf scars at concentrations of 105 and 108 CFU ml−1, respectively. In particular, the application of 6,000 arbitrary units (AU) of crude bacteriocin (dialyzed ammonium sulfate precipitate of culture supernatant) ml−1 at the inoculated V-shaped slits and leaf scars resulted in the formation of knots with weight values reduced by 81 and 51%, respectively, compared to the control, depending on the strains and inoculation method used. Crude bacteriocin (6,000 AU ml−1) was also effective in controlling the multiplication of epiphytic populations of the pathogen. In particular, the bacterial populations recovered after 30 days were at least 350 and 20 times lower than the control populations on twigs and on leaves, respectively. These results suggest that bacteriocin from P. syringae pv. ciccaronei can be used effectively to control the survival of the causal agent of olive knot disease and to prevent its multiplication at inoculation sites.

Isomerization of Peroxidizing Thiadiazolidine Herbicides Is Catalyzed by Glutathione S-Transferase

Thiadiazolidine-converting activity (isomerase), detected in a 45-75% ammonium sulfate precipitate from corn seedlings extracts, was purified by chromatography on hydroxyapatite and by anion exchange on Mono Q Sepharose. Two fractions 1 and 2 with isomerase activity were separated on Mono Q by combination of a stepwise elution and continuous salt gradient; fraction 2 eluting at higher salt concentrations was found the most active. Total activity could be enhanced by treatment of seedlings with naphthalic anhydride. Both fractions containing isomerase activity were further purified by glutathione-(GSH) agarose affinity chromatography and characterized by their specificity for different thiadiazolidines. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and gel filtration revealed that the isomerase of fraction 2 consists either of a homodimer or a heterodimer of two proteins with apparent molecular weights of 28 and 31 kDa, respectively. The protein pattern as well as the strict dependence of activity on thiol groups (GSH or dithiothreitol) suggested a glutathione Stransferase (GST) catalyzing the thiadiazolidine conversion. Further evidence was obtained by measuring reactions specific for GSTs in both purified fractions, namely the conjugating activity for l-chloro-2,4-dinitrobenzene (CDNB ). atrazine and metazachlor. While no atrazine turnover was found, metazachlor and CDNB conjugation occurred rapidly. Both fractions differed in their activities to several GST substrates with fraction 2 being more effective in metazachlor but less active in C DN B conjugation. Inhibitors specific for GST-catalyzed reactions also inhibited thiadiazolidine conversion confirming that isomerizing activity is attributed to a GST form. We conclude that GST isoforms with different affinities towards thiadiazolidines have been isolated. CDNB activity, molecular weight, the protein pattern on SDS-PAGE as well as the amino acid sequence of one of its polypeptides suggest that fraction 1, less active in thiadiazolidine isomerization, is identical to GST I. The second peptide of this fraction was resistant to Edman degradation probably due to N-terminal blockage. The properties of the high isomerase activity found in fraction 2 are in agreement with characteristics of a GST previously termed as isoform II.

STUDIES ON PHOSPHATASES IN HUMAN PLATELETS

Activation of platelets by various agonists has been ascribed to be associated with phosphorylation and dephosphorylation of specific proteins such as 20K and 47K polypeptide. Although protein kinases such as myosin light chain kinase and C kinase have been extensively studied, little information is currently available on platelet phosphatases, which may play a crucial role in the regulation of stimulus-linked protein phosphorylation. Thereby, the present study was conducted to know some characters of platelet phosphatases. Glycerol loaded platelets prepared from human platelet concentrates were subjected to osmotic lysis in 20 mM HEPES-NaOH buffer containing 5 mM EDTA, 0.5 mM dithio-threitol and various protease inhibitors and a soluble fraction was obtained by centrifugation, The activity of phosphatase was assayed at pH 7.35, using paranitrophenylphosphate as a substrate. Leupeptin and EDTA were added to the reaction mixture to avoid proteolytic attack to the enzyme. The neutral phosphatase was partially purified from the soluble fraction by a combination of ammonium sulfate fractionation and column chromatographies. Five distinct peaks with neutral phosphatase activity were obtained by a linear gradient elution ( 0−0.5 M KCl ) in DEAE Sepharose CL-6B of 0−60 % ammonium sulfate precipitate. The phosphatase activity of one peak eluted at 0.2M KCl was maximum at pH below 6, which was considered to be acid phosphatase, and the remaining four peaks' optimal pH was between 7.0−7.5. These four peaks were termed as PH-I (passed through fraction), PH-II (0.1M KCl), PH-III (0.25M KCl) and PH-IV (0.3M KCl). The respective peak was eluted as a single peak on Ultrigel AcA 34 and the molecular weight was estmated as follows; I-55K, II-40K, III-55K, IV-37K. PH-I − II were active in the presnce of EDTA and were not affect ed by divalent cations (Mg++ , Mn++ , Ca++ ) , whereas PH-III was highly dependent upon Mg++. The activity of PH-IV was completely dependent on Mn++. From these observations, the following conclusions were obtained; (1) Human platelets contain four species of neutral phosphatases, in addition to acid phosphatase. (2) Each neutral phosphatase is distincive by molecular weight and requirement of divalent cations.

Further studies on the corticosteroid-receptor system of the nasal gland of the domestic duck (Anas platyrhynchos)

The interaction of tritiated corticosterone with the nasal gland corticosterone receptor was investigated. Kinetic studies have shown that the association of [3H]corticosterone–receptor followed second-order reaction kinetics and the dissociation of the ligand from the receptor became "pseudo" first order in the presence of large excess of radioinert steroids at 0, 15, 25, and 35 °C. Similar data were obtained with an ammonium sulfate precipitate of the cytosol. Dissociation rate constants varied from 10−5 to 10−3 s−1 and the association rate constants varied from 0.5 × 104 to 3.8 × 105 M−1∙s−1, depending on the reaction temperature and the cytoplasmic receptor preparation. Equilibrium dissociation constants were in 10−8–10−9 M range. Equilibrium dissociation constants, calculated from kinetic data (kd/ka), showed a marked temperature dependence, while those obtained by saturation analysis varied much less with the reaction temperature. Data obtained in these experiments were used to calculate some thermodynamic parameters of the binding of corticosterone to the cytoplasmic receptor. The enthalpy of dissociation was 101.5 and 79.4 kJ∙mol−1 and the entropy of dissociation was 200 and 280 J∙mol−1∙degree−1 for the crude cytoplasmic receptor and the ammonium sulfate precipitate, respectively. From the equilibrium dissociation constants, the enthalpy and entropy of the equilibrium binding was calculated. Polynomial fitting of Ka values versus 1/T yielded enthalpy (ΔH) values from −0.9 to −88.8 kJ∙mol−1, depending on the nature of the receptor preparation. Entropy values were negative for kinetically derived equilibrium association constants from the crude cytosol at all temperatures and for 0 and 15 °C for the precipitate. Entropy values were positive for Ka values obtained from kinetic rates at 25 and 35 °C and for Ka's calculated from saturation analysis. Further experiments with the precipitate confirmed our previous contention that the nasal gland cytoplasmic corticosterone receptor metabolized the bound ligand to 11-dehydrocorticosterone, though the receptor preparation was corticosterone specific. The following hydrodynamic parameters were obtained on the binding macromolecule: molecular weight, 316 000; s20,w, 8.0; Stokes radius (rs), 77.3 Å (1 Å = 0.1 nm); total frictional ratio (f/f0), 1.71. The labeled receptor preparation translocated to homologous nuclear binding sites following heat activation and, at the nuclear binding sites, the ligand was almost exclusively in its oxidized form. Measurement of the nuclear steroid–receptor complex by exchange assay with [3H]corticosterone confirmed the presence of nuclear binding sites. From these studies, it was concluded that the nasal gland of the duck contains specific, glucocorticoid-type corticosterone receptors and that the effector steroid is probably 11-dehydrocorticosterone or a critical mixture of these two steroids, with the oxidized form predominating.

A rapid method for the determination of CTP and phosphocholine in rat liver and baby hamster kidney 21 cells

A rapid and sensitive assay for CTP and phosphocholine was required for us to determine the concentration of these compounds in tissues and cell cultures. Such a procedure was devised with CTP:phosphocholine cytidylyltransferase, an enzyme which is highly specific for CTP and phosphocholine. The 0–22% ammonium sulfate precipitate of a cytosolic extract from rat liver was used as the source of the enzyme. The amount of CTP in an extract was estimated by the conversion of [3H]phosphochoiine to 3H-labelled CDP-choline. Similarly, the concentration of phosphocholine was estimated by the formation of 3H-labelled CDP-choline from 3H-labelled CTP. The conversion of CTP and phosphocholine to CDP-choline was 90% when inorganic pyrophosphatase was added to the incubations. The formation of CDP-choline was linear between 1 and 10 nmol of CTP or phosphocholine. The concentration of CTP was determined in rat liver (62 nmol/g wet weight) and baby hamster kidney 21 (BHK-21) cells (161 nmol/g wet weight). The concentration of phosphocholine in rat liver was 1.16 μmol/g wet weight whereas in BHK-21 cells it was much less (69 nmol/g wet weight). By this procedure, it may be possible to establish the importance of CTP and phosphocholine in the control of phosphatidylcholine biosynthesis.

Detection and Partial Purification of a Natural Heparin Inhibitor

A naturally occurring heparin inhibitor has been detected in the mucosa of the fresh hog small intestine and has been partially purified. After the homogenized mucosa was extracted with Tris buffer overnight (3°) and the resulting supernatant was fractionated with ammonium sulfate, a large quantity of antiheparin activity was detected in the ammonium sulfate precipitate. This precipitate contains antiheparin activity with a specific activity of 0. 68 unit/mg of protein. Therefore, each hog small intestine contains an amount of this inhibitor enough to inhibit approximately 20, 000 units of heparin. Further purification of this heparin inhibitor was carried out by the technique of heparin affinity chromatography (covalently linked the heparin by the cyanogen bromide procedure). Eluted by a controlled NaCl and buffered gradient at 3°, the chromatogram contains a major peak and a minor peak. Antiheparin activity was located in the minor peak and has a specific activity of 9·7 units/mg of protein. Thus, we have achieved a 14-fold purification of this heparin inhibitor. This partially purified protein inhibits heparin stoichiometrically. Further experiments to purify this heparin inhibitor are in progress. This naturally occurring heparin inhibitor probably has an important biological function in balancing the action of heparin which is an important factor in maintaining Mood fluidity.

The Conjugated Estrogens. II. A New Method for Isolating Estriol Monoglucosiduronide from Normal Human Pregnancy Urine

Estriol glucosiduronide has been prepared from normal pregnancy urine both as sodium salt and as the free carbonyl forms by a new and simple five-step procedure: (i) precipitation by ammonium sulfate of the conjugated estrogens from the urine; (ii) preparation of a methanol–acetone (M–A) solution of the conjugated estrogens from the ammonium sulfate precipitate; (iii) filtration through columns of Sephadex G 25 of the combined M–A residues from large batches of urine, which yielded the starting material for the work presented in this paper, namely peak four of the six estrogen peaks thus obtained; (iv) the conversion to the carbonyl form by a "Kellie" extraction at pH 2.0–2.5; and (v) crystallization of the acid or of its sodium salt from the semi-crystalline residue.