Correlation between polymerase chain reaction analysis of the histidine biosynthesis operon, randomly amplified polymorphic DNA analysis and phenotypic characterization of dairy Lactococcus isolates

A collection of 32 lactococcal strains isolated from raw milk in the Camembert RDO (registered designation of origin) area were phenotypically and genotypically characterized. As expected for environmental isolates, all strains had a Lactococcus lactis subsp. lactis phenotype. The strains were then genotypically identified by the randomly amplified polymorphic DNA (RAPD) technique, using reference strains of lactococci. Two major clusters were identified containing the two subspecies lactis and cremoris. The subspecies lactis cluster could be divided into five subgroups whereas there was a high coefficient of similarity between all strains in the subspecies cremoris cluster. This RAPD classification was then compared with that of a traditional PCR assay using L.lactis species-specific primers corresponding to part of the histidine biosynthesis operon. The two subspecies were differentiated by the size of the fragment amplified (about 200 bp longer for subspecies cremoris). Unlike preliminary phenotypic assignments, the results of PCR experiments corroborated the genotypic identification of the lactococcal strains by RAPD allowing the technique to be reconsidered on the basis of its taxonomic efficiency. Received: 14 May 1998 / Accepted: 3 September 1998     

Chemical cleavage of bovine beta-lactoglobulin by BNPS-skatole for preparative purposes: comparative study of hydrolytic procedures and peptide characterization

A comparative study of various procedures for tryptophanyl peptide bond cleavage by BNPS-skatole [2-(2-nitrophenyl)-3-methyl-3-bromoindolenine] was carried out on native and on reduced and alkylated bovine -lactoglobulin (BLG). The reaction yield and the composition of the derived products were studied in acetic acid, trifluoroacetic acid (TFA), and ethanol/TFA. For BNPS-skatole removal, extraction by water or ethyl ether was compared with dialysis and gel filtration. The three expected peptides (1–19, 20–61, 62–162) and incomplete cleaved fragments (1–61, 20–162) were separated and characterized by electrophoresis, reverse-phase high-performance liquid chromatography, and mass spectrometry. The highest hydrolysis yield (67.4%) occurred with native BLG cleaved in 88% acetic acid at 47°C for 60 min. Subsequent water extraction and gel filtration led to total recovery of the material, but reagent elimination was only quantitative after gel filtration. Cleavage specificity was ensured by mass spectrometry and the amino acid composition of peptides 1–19 and 62–162. The chemical side reactions identified are discussed.     

Trichothecenes and zearalenone production by fusarium species isolated from Argentinean black beans

Isolates of Fusarium species obtained from freshly harvested bean grains for human consumption collected from different Argentinean regions, were investigated for their ability to biosynthesise trichothecenes and zearalenone either on rice grains or beans. Low incidence of toxigenic fungi was observed. These mycotoxigenic species produced several toxins when grown on rice but none or little amount when cultured on beans. The results of this report suggest that contamination of Argentinean beans with Fusarium mycotoxins will not be common and therefore people would be at low mycotoxicosis risk through consumption of beans.     

Proteomics application exercise of the Swiss Proteomics Society: report of the SPS'02 session

After the success of the mass spectrometry (MS) round table that was held at the first Swiss Proteomics Society congress (SPS'01) in Geneva, the SPS has organized a proteomics application exercise and allocated a full session at the SPS'02 congress. The main objective was to encourage the exchange of expertise in protein identification, with a focus on the use of mass spectrometry, and to create a bridge between the users' questions and the instrument providers' solutions. Two samples were sent to fifteen interested labs, including academic groups and MS hardware providers. Participants were asked to identify and partially characterize the samples. They consisted of a complex mixture of peptide/proteins (sample A) and an almost pure recombinant peptide carrying post-translational modifications (sample B). Sample A was an extract of snake venom from the species Bothrops jararaca. Sample B was a recombinant and modified peptide derived from the shrimp Penaeus vannamei penaeidin 3a. The eight labs that returned results reported the use of a wide range of MS instrumentation and techniques. They mentioned a variety of time and manpower allocations. The origin of sample A was generally identified together with a number of database protein entries. The difficulty of the sample identification lay in the incomplete knowledge of the Bothrops species genome sequence and is discussed. Sample B was generally and correctly identified as penaeidin. However, only one group reported the full primary structure. Interestingly, the approaches were again varied and are discussed in the text.     

An association between the acute phase response and patterns of antigen induced T cell proliferation in juvenile idiopathic arthritis

The aim of this research was to determine whether all memory T cells have the same propensity to migrate to the joint in patients with juvenile idiopathic arthritis. Paired synovial fluid and peripheral blood mononuclear cell proliferative responses to a panel of antigens were measured and the results correlated with a detailed set of laboratory and clinical data from 39 patients with juvenile idiopathic arthritis. Two distinct patterns of proliferative response were found in the majority of patients: a diverse pattern, in which synovial fluid responses were greater than peripheral blood responses for all antigens tested; and a restricted pattern, in which peripheral blood responses to some antigens were more vigorous than those in the synovial fluid compartment. The diverse pattern was generally found in patients with a high acute phase response, whereas patients without elevated acute phase proteins were more likely to demonstrate a restricted pattern. We propose that an association between the synovial fluid T cell repertoire and the acute phase response suggests that proinflammatory cytokines may influence recruitment of memory T cells to an inflammatory site, independent of their antigen specificity. Additionally, increased responses to enteric bacteria and the presence of αEβ7 T cells in synovial fluid may reflect accumulation of gut associated T cells in the synovial compartment, even in the absence of an elevated acute phase response. This is the first report of an association between the acute phase response and the T cell population recruited to an inflammatory site.     

An Ecological Study of Lactococci Isolated from Raw Milk in the Camembert Cheese Registered Designation of Origin Area

The genetic diversity of lactococci isolated from raw milk in the Camembert cheese Registered Designation of Origin area was studied. Two seasonal samples (winter and summer) of raw milk were obtained from six farms in two areas (Bessin and Bocage Falaisien) of Normandy. All of the strains analyzed had a Lactococcus lactis subsp. lactis phenotype, whereas the randomly amplified polymorphic DNA (RAPD) technique genotypically identified the strains as members of L. lactis subsp. lactis or L. lactis subsp. cremoris. The genotypes were confirmed by performing standard PCR with primers corresponding to a region of the histidine biosynthesis operon. The geographic distribution of each subspecies of L. lactis was determined; 80% of the Bocage Falaisien strains were members of L. lactis subsp. lactis, and 30.5% of the Bessin strains were members of L. lactis subsp. lactis. A dendrogram was produced from a computer analysis of the RAPD profiles in order to evaluate the diversity of the lactococci below the subspecies level. The coefficient of similarity for 117 of the 139 strains identified as members of L. lactis subsp. cremoris was as high as 66%. The L. lactis subsp. lactis strains were more heterogeneous and formed 10 separate clusters (the level of similarity among the clusters was 18%). Reference strains of L. lactis subsp. lactis fell into 2 of these 10 clusters, demonstrating that lactococcal isolates are clearly different. As determined by the RAPD profiles, some L. lactis subsp. lactis strains were specific to the farms from which they originated and were recovered throughout the year (in both summer and winter). Therefore, the typicality of L. lactis subsp. lactis strains was linked to the farm of origin rather than the area. These findings emphasize the significance of designation of origin and the specificity of “Camembert de Normandie” cheese.     

Purification and Characterization of Anti-Listeria Compounds Produced by Geotrichum candidum

Geotrichum candidum can produce and excrete compounds that inhibit Listeria monocytogenes. These were purified by ultrafiltration, centrifugal partition chromatography, thin-layer chromatography, gel filtration, and high-pressure liquid chromatography, and analyzed by liquid chromatography-mass spectrometry, infrared spectrometry, nuclear magnetic resonance spectrometry, and optical rotation. Two inhibitors were identified: d-3-phenyllactic acid and d-3-indollactic acid.

Contamination by Listeria has become a problem over the past 20 years in many parts of the world. The ubiquitous nature of Listeria monocytogenes, its capacity to multiply at refrigeration temperatures, its thermal tolerance (11), and its resistance to relatively low pH (it can multiply at pH 5.3 and 4°C and at pH 4.39 and 30°C) (5), together with its tolerance of high salt concentrations (4, 18), make controlling this potentially pathogenic microorganism in food products difficult. This bacterium has been incriminated in several cases of food poisoning (2, 10, 19). At risk are the immunodepressed, the old, pregnant women, fetuses, and newborn babies. Several groups have worked on biological control. As a result, many bacteriocins, which inhibit the growth of L. monocytogenes, have been isolated, purified, and characterized (12, 13, 16, 18). We have worked with Geotrichum candidum, a yeast-like member of the natural milk flora that is used as a maturing agent for soft and hard cheeses. In an extensive study carried out in 1984 (7), the interactions between G. candidum and the microflora in cheeses were examined. G. candidum inhibited the growth of gram-negative bacteria, gram-positive bacteria, and fungi (6). We recently showed (3) that G. candidum inhibits the growth of L. monocytogenes on both solid and liquid media (a bacteriostatic effect). The inhibitors are stable over a wide pH range and can be heated to 120°C for 20 min. The present report describes the purification and characterization of compounds responsible for this antibacterial action.Microorganisms, culture conditions, and detection of inhibitory activity.The strain of G. candidum used came from the collection of the Caen University Food Microbiology Laboratory, Caen, France (UCMA G91) and was initially isolated from a cheese, Pont l’Evêque. One percent of a preculture (optical density at 620 nm [Milton Roy Spectronic 301; Bioblock Scientific, Illkirch, France] of 0.7 [10⁷ arthrospores or hyphae/ml]) of G. candidum was grown in a fermentor (20 liters; Biolafitte type PI) in 15 liters of Trypticase soy broth (30 g/liter; Biomerieux, Marcy l’Etoile, France) with yeast extract (6 g/liter; AES, Combourg, France) (TSBYE) buffered to pH 6.3 with 0.1 M citrate-0.2 M phosphate. The culture was stirred at 300 rpm for 64 h at 25°C under a pressure of 0.2 bar and was then filtered through a 1,000-Da cut-off membrane by tangential ultrafiltration (Sartorius, Palaiseau, France) under a pressure of 2 bars. The resulting ultrafiltrate was sterilized by passage through a capsule (Sartorius) containing 0.45-μm- and 0.2-μm-pore-size membranes.The inhibition of L. monocytogenes was checked at each purification step by the agar diffusion well assay (3). Antimicrobial activity was estimated by measuring the diameter of the inhibitory halo on two right-angle axes (average of two plates). The strain of L. monocytogenes (UCMA L205) (serovar 1/2a; Centre National de Référence des Listeria, Nantes, France) and lysovar 1652 (Institut Pasteur, Paris, France) came from the laboratory collection and was isolated from milk. The initial lyophilized ultrafiltrate (900 mg/ml) gave a halo diameter of 36 ± 0.7 mm in the inhibition assay.Purification.Samples of ultrafiltrate (20 μl) were spotted on thin-layer chromatography (TLC) plates (silica gel, 10 by 5 cm, 0.25 mm thick, 60 F₂₅₄; Merck, Darmstadt, Germany), with 20 μl of TSBYE for controls, and eluted by vertical chromatography with a butanol-acetic acid-water (40:10:20 [vol/vol/vol]) solvent system. The bands were examined under UV light (254 nm) or after treatment with Ehrlich’s reagent. Four well-separated bands were found (R_fs, 0.11 ± 0.04; 0.41 ± 0.04; 0.7 ± 0.03; and 0.86 ± 0.03), but only the band with an R_f of 0.7 ± 0.03 differed from that of control preparations (TSBYE not containing G. candidum). The microbiological bioautography test (1) confirmed the presence of the inhibitor in the band with an R_f of 0.7. Lyophilized ultrafiltrate was subjected to centrifugal partition chromatography (Sanki 1000 Engineering Ltd.; EverSeiko, Tokyo, Japan) in butanol-acetic acid-water (40:10:50 [vol/vol/vol]). Partitioning was carried out under the following conditions: ascending mode, 1,200 rpm; flow rate, 3 ml/min; pressure, 40 bars. An aliquot of material (3 g) previously equilibrated with the solvent system was injected into the separatus via a 12-ml injection loop. Fractions (10 ml each) were collected and evaporated to dryness in a SpeedVac (Jouan RC 1022, Saint Herblain, France). The dried extracts of certain fractions were taken up in 800 μl of water and brought to pH 5.6 with 0.2 M NaOH. A total of 10 mg of pooled fractions with an R_f close to 0.7 and showing L. monocytogenes inhibitory activity (36 ± 0.7 mm for a solution of 38 mg/ml) was taken up in 250 μl of methanol-water (50:50 [vol/vol]) and automatically deposited (Camag Linomat) on a 10- by 20-cm TLC plate (silica gel, 0.25 mm thick, 60 F₂₅₄; Merck). The plate was developed with butanol-acetic acid-water (40:10:20 [vol/vol/vol]) and examined under UV light. Bands with an R_f of 0.7 were scraped off and placed in methanol. The silica was washed several times and removed by centrifugation and filtration through a 0.2-μm-pore-size filter. An aliquot (20 μl) was spotted on a small silica TLC plate to confirm elution of the solute by the methanol solvent. The purity of the band with an R_f of 0.7 was confirmed by high-pressure liquid chromatography (HPLC) coupled with a photodiode array detector at 206 and 222 nm (solvent A: 0.1% formic acid in water; solvent B: CH₃CN-H₂O [95:5] plus 0.1% formic acid) on a C₁₈ Grom-Sil ODS2 column (4.6 by 30 mm; particle size, 1.5 μm; Grom Analytic, Herrenberg, Germany) at the flow rate of 1 ml/min. Inhibitory activity was assessed as above. Preparative TLC indicated that the band with an R_f of 0.7 contained two components, one eluting at 4.5 min on HPLC (peak 1) and the other at 5.5 min (peak 2). The latter fraction gave an inhibitory halo of 36 ± 0.7 mm at a concentration of 20 mg/ml (a 45-fold purification over the ultrafiltrate). The material from preparative TLC (40 mg in 200 μl of methanol-water) was placed on a column of Sephadex LH20 (1 m by 1 cm; Pharmacia), and the column was eluted with methanol-water at 12 ml h⁻¹. Fractions (1 ml each) were collected and examined by HPLC to determine the material in each fraction. This final purification on Sephadex LH20 gave two peaks, with two-thirds of the eluate at peak 1 and one-third at peak 2 (Fig. ​(Fig.1).1). As the concentration for peak 2 was very low, only the inhibitory activity for peak 1 was assayed. A concentration of 20 mg/ml gave a halo diameter of 26 ± 0.7 mm. Open in a separate windowFIG. 1Reverse-phase liquid chromatography (HPLC) of inhibitory compounds of G. candidum after purification by centrifugal partition chromatography, preparative TLC, and Sephadex LH20 gel filtration. Column, C₁₈ Grom-Sil ODS2 column (4.6 by 30 mm; particle size, 1.5 μm; Grom Analytic). Eluent: solvent A (0.1% formic acid in water), solvent B (CH₃CN-H₂O [95:5] plus 0.1% formic acid). Flow rate, 1 ml/min. (a) Product 1 (detection at 206 nm); (b) product 2 (detection at 222 nm).

Characterization.

The pooled fractions were run on HPLC with a Grom-Sil ODS2 column coupled to a mass detector (Sciex Api III, triple quadrupole; Thornhill, Canada). Product 1, analyzed by desorption and chemical ionization, gave a signal at an m/z of 184 for (M⁺ NH₄)⁺ on desorption and chemical ionization and thus had a mass of 166. Product 2 was analyzed by ion spray and gave a signal at an m/z of 297 (M + 4 Na)⁺ for a mass of 205. Spectra were determined in a Nicolet model 60 SXR FT-IR. Samples were dissolved in dimethyl sulfoxide, and the ¹H and ¹³C resonances were measured in a Brucker spectrometer at 200 and 400 Hz, respectively. The purified material was taken up in methanol, and the isomeric form of the substance(s) inhibiting L. monocytogenes was determined in a Perkin-Elmer model 341 polarimeter. Two inhibitors were identified (Fig. ​(Fig.2);2); product 1 was 2-hydroxy-3-phenylpropanoic acid (phenyllactic acid, mass 166), and product 2 was 2-hydroxy-3-indolpropanoic acid (indollactic acid, mass 205). The rotation of polarized light showed that the phenyllactic acid produced by G. candidum was the d form. The spectrum properties of the isolated compounds are identical to those of authentic commercial compounds (Sigma Chemical Co., St. Louis, Mo.). d-Phenyllactic acid can be purchased from Aldrich (product no. 37 690-6), and dl-indollactic acid is available from Sigma (catalog no. I2875). Inhibitory activity with commercial compounds showed that dl-phenyllactic acid (Sigma catalog no. P7251) was a stronger inhibitor of Listeria than dl-indollactic acid (34 and 26 ± 0.7 mm for 187 mM, respectively) and that the d form of phenyllactic acid was more active (38 mm for 120 mM) than the l form (Aldrich 11, 306-9, 30 mm for 120 mM). The samples were taken up in methanol-water (50:50 [vol/vol]) and brought to pH 5.6 (Table ​(Table1).1). Open in a separate windowFIG. 2Structure of two inhibitory compounds of G. candidum characterized by LC-mass spectrometry, infrared spectrometry, nuclear magnetic resonance spectrometry, and optical rotation.TABLE 1Anti-Listeria activity of phenyllactic acid and indollactic acid^a

Protocol of a prospective study on the diagnostic value of transcranial duplex scanning of the substantia nigra in patients with parkinsonian symptoms

Background  

Parkinson's disease (PD) is the second most common neurodegenerative disorder. As there is no definitive diagnostic test, its diagnosis is based on clinical criteria. Recently transcranial duplex scanning (TCD) of the substantia nigra in the brainstem has been proposed as an instrument to diagnose PD. We and others have found that TCD scanning of substantia nigra duplex is a relatively accurate diagnostic instrument in patients with parkinsonian symptoms. However, all studies on TCD so far have involved well-defined, later-stage PD patients, which will obviously lead to an overestimate of the diagnostic accuracy of TCD.     

Bioactive poly(ethylene terephthalate) fibers and fabrics: grafting, chemical characterization, and biological assessment

The grafting of poly(sodium styrene sulfonate) (pNaSS) onto ozone-treated poly(ethylene terephthalate) (PET) fabric surfaces was characterized by X-ray photoelectron spectroscopy and toluidine blue colorimetry. Significant amounts of pNaSS were grafted over the range of experimental conditions examined in this study (30-120 min of ozonation, reaction at 65 or 70 degrees C, and reaction times up to 240 min). Within these ranges the amount of grafted pNaSS increased with both ozonation time and reaction temperature. The amount of grafted pNaSS increased over the first 60 min of reaction, then remained relatively constant from 60 to 240 min. For the biological experiments pNaSS-grafted samples were prepared with 30 min of ozonation and 60 min of reaction at a grafting temperature of 70 degrees C. The ozonation time was limited to 30 min to minimize any possible degradation of the PET fabrics by the ozonation treatment. The pNaSS-grafted PET surface adsorbed a factor of 4 more compared to the nongrafted surfaces. The strength of fibroblast adhesion was an order of magnitude higher on pNaSS-grafted PET fabrics compared to that on nongrafted PET fabrics. This difference in the cell attachment was correlated to the cell spreading, which was better and more homogeneous on the grafted fibers compared to the nongrafted fibers. Fibroblasts adhered more strongly on surfaces precoated with normal human plasma compared to surfaces precoated with 10% fetal calf serum in Dulbecco's modified Eagle's medium.