A portable electrochemical magnetoimmunosensor for detection of sulfonamide antimicrobials in honey.

A new electrochemical magnetoimmunosensor (EMIS) has been developed for the screening of residues of sulfonamide antimicrobials in honey samples. The immunosensor is able to detect up to ten different sulfonamide congeners at levels below the action points established in some European countries (25 µg kg(-1)) after a hydrolysis step in which the sulfonamides are released from the corresponding conjugates formed in samples of this type. In spite of the complexity of the sample after the hydrolysis procedure, the EMIS could perform quantitative measurements, directly in these samples, without any additional sample cleanup or extraction step. For example, sulfapyridine, used as a reference, can be detected in hydrolyzed honey with a limit of detection (IC90) of 0.1 ± 0.03 µg kg(-1). Considering that the use of antibiotics for bee treatment is prohibited in the European Union, the immunosensor presented here could be an excellent screening tool. Moreover, several samples can be processed in parallel, which facilitates the analysis, reducing the necessity to use more costly confirmatory methods for just screening. As a proof of concept, a set of blind honey samples (spiked and incurred) were analyzed and the results were compared with those obtained by high-performance liquid chromatography-tandem mass spectrometry, demonstrating the potential of the EMIS as a screening tool.

Rapid immunochemical analysis of the sulfonamide-sugar conjugated fraction of antibiotic contaminated honey samples.

A rapid high-throughput immunochemical screening (HtiS) procedure for the analysis of the sulfonamide (SA)-sugar conjugated fraction of antibiotic contaminated honey samples has been developed. Studies performed with this matrix have indicated that sulfonamide antibiotics are conjugated to sugars rapidly and quantitatively, providing samples with very low SA immunoreactivity. Therefore, sulfonamides must be first released before the analysis, and for this purpose, a simple and fast sample preparation procedure has been established consisting of hydrolyzing the sample for 5 min, adjusting the pH and buffering the sample prior to the immunochemical analysis. Under these conditions, honey samples could be directly analyzed without any additional sample treatment, other than dilution. Recovery values of the whole analytical procedure were greater than 85%. The analysis of the same samples without the hydrolysis provided recovery values below 5%. Selectivity studies performed in hydrolyzed honey samples revealed that nine relevant sulfonamide antibiotics can be detected with limit of detection (LOD) values below the action limits established by some EU countries (Belgium, 20 µg kg(-1), United Kingdom or Switzerland, 50 µg kg(-1)).

An additional aromatic interaction improves the thermostability and thermophilicity of a mesophilic family 11 xylanase: structural basis and molecular study.

In a general approach to the understanding of protein adaptation to high temperature, molecular models of the closely related mesophilic Streptomyces sp. S38 Xyl1 and thermophilic Thermomonospora fusca TfxA family 11 xylanases were built and compared with the three-dimensional (3D) structures of homologous enzymes. Some of the structural features identified as potential contributors to the higher thermostability of TfxA were introduced in Xyl1 by site-directed mutagenesis in an attempt to improve its thermostability and thermophilicity. A new Y11-Y16 aromatic interaction, similar to that present in TfxA and created in Xyl1 by the T11Y mutation, improved both the thermophilicity and thermostability. Indeed, the optimum activity temperature (70 vs. 60 degrees C) and the apparent Tm were increased by about 9 degrees C, and the mutant was sixfold more stable at 57 degrees C. The combined mutations A82R/F168H/N169D/delta170 potentially creating a R82-D169 salt bridge homologous to that present in TfxA improved the thermostability but not the thermophilicity. Mutations R82/D170 and S33P seemed to be slightly destabilizing and devoid of influence on the optimal activity temperature of Xyl1. Structural analysis revealed that residues Y11 and Y16 were located on beta-strands B1 and B2, respectively. This interaction should increase the stability of the N-terminal part of Xyl1. Moreover, Y11 and Y16 seem to form an aromatic continuum with five other residues forming putative subsites involved in the binding of xylan (+3, +2, +1, -1, -2). Y11 and Y16 might represent two additional binding subsites (-3, -4) and the T11Y mutation could thus improve substrate binding to the enzyme at higher temperature and thus the thermophilicity of Xyl1.

Sequence, overproduction and purification of the family 11 endo-beta-1,4-xylanase encoded by the xyl1 gene of Streptomyces sp. S38.

The xyl1 gene encoding the Xyl1 xylanase of Streptomyces sp. strain S38 was cloned by screening an enriched DNA library with a specific DNA probe and sequenced. Three short 5 bp -CGAAA- sequences are located upstream of the Streptomyces sp. S38 xyl1 gene 105, 115 and 250 bp before the start codon. These sequences, named boxes 1, 2 and 3, are conserved upstream of the Actinomycetales xylanase genes and are specifically recognized by a DNA-binding protein (Giannotta et al., 1994. FEMS Microbiol. Lett. 142, 91-97) and could be probably involved in the regulation of xylanase production. The Xyl1 ORF encodes a 228 residue polypeptide and the Xyl1 preprotein contains a 38 residue signal peptide whose cleavage yields a 190 residue mature protein of calculated M(r) = 20,585 and basic pI value of 9.12. The molecular mass of the produced and purified mature protein determined by mass spectrometry (20,586 +/- 1 Da) and its pI (9.8) agree with these calculated values. Its N-terminal amino-acid sequence confirmed the proposed cleavage site between the signal peptide and the mature protein. Comparisons between Xyl1 and the 62 other xylanases belonging to family 11 allowed the construction of a phylogenetic tree and revealed its close relationship with Actinomycetales enzymes. Moreover, nine residues were found to be strictly conserved among the 63 xylanases.

The precursor of the Streptomyces R61 DD-peptidase containing a C-terminal extension is inactive.

The Streptomyces R61 DD-peptidase gene encodes a 26-residue C-terminal extension which is not found in the mature protein. When the gene was expressed in Escherichia coli, the extension was not cleaved and the precursor protein was not enzymatically active. It also reacted with penicillins significantly more slowly than the mature protein. The introduction of a 'stop' codon after that corresponding to the C-terminal residue of the mature protein resulted in the production of an active protein in the periplasm of E. coli.

Diversity of the mechanisms of resistance to beta-lactam antibiotics.

The sensitivity of a bacterium to beta-lactam antibiotics depends upon the interplay between 3 independent factors: the sensitivity of the essential penicillin-binding enzyme(s), the quantity and properties of the beta-lactamase(s) and the diffusion barrier that the outer-membrane of Gram-negative bacteria can represent. Those three factors can be modified by mutations or by the horizontal transfer of genes or portions of genes.

Degradation products of the process of thermal recovery of copper from lamina scraps in lab-scale fluidized bed reactor.

This paper presents experimental results dealing with a process for recovering copper in the scrap composite materials issued from electronic laminas industry. This environment-friendly process consists in the thermal treatment of scrap in a fluidized bed whose particles fix the harmful gases emitted by the organic glue gasification. A series of experiments was carried out in a thermobalance coupled to FTIR spectrometer and GC/MS with small lamina samples. These experiments demonstrated the thermal behavior of scrap composite materials, and identified the major degradation reaction gases. A series of experiments was performed with bigger scrap samples hung in a laboratory-scale fluidized bed coupled to FTIR and MS, at 350 degrees C; the results confirmed those obtained in thermobalance. Experiments showed that a residence time lasting less than 5 min is sufficient to recover the metallic copper, and exhaust gases are not harmful.

Restoration of images degraded by atmospheric turbulence by a least-squares method and a Markov process.

We present a two-step deconvolution method for restoring images degraded by atmospheric turbulence. The first step is linear space-invariant filtering, and the second step is a nonhomogeneous Markov process. This nonhomogeneous method preserves the discontinuities of the original image better than the homogeneous method does.

[Complications of transurethral resection in the treatment of benign prostatic hypertrophy. Apropos of a series of 1180 adenomas--1976-1986]. / Complications de la résection transurétrale dans la cure de l'hypertrophie prostatique bénigne. A propos d'une série de 1 180 adénomes--1976-1986.

This was a homogeneous series treated by the same surgeon between January 1977 and December 1986. During this period, out of 1,806 certico-prostatic obstacles, 1,180 were treated by resection of a prostatic adenoma with a mean age of 68,3 years and a range of 37 to 94 years. The surgical indications fell into four groups: dominant dysuria, dominant pollakiuria, complicated adenomas and patients with a single past episode of acute urinary retention. Resection was performed with a direct current (Iglésias) essentially with sterile demineralized water. General anesthesia was used in 2/3 of the cases and loco-regional anesthesia in 1/3 of the cases. A traditional resection technique was used, and postoperative bladder drainage used a Mercier 24 catheter. Continuous lavage was not performed. Duration of resection was 10 to 120 minutes with a mean of 39 minutes. The resected weight ranged between 8 and 130 grams with a mean of 24.2 grams. Duration of the indwelling catheter decreased from 5 days in 1976 to 24 or 48 hours in 1982. Duration of hospitalization decreased from 12 to 7 days, and down to 5 days today. Complications were uncommon: 2 deaths (0.17%) due to acute pancreatitis and mesenteric infarction; 1 perforated bladder; 4 septicemia with shock; 2 acute renal failures requiring dialysis; 5 pulmonary emboli; 3 hemiplegia; 1 acute nonfatal pancreatitis. Only 50 patients (4.2%) required transfusions. There was no permanent urinary incontinence but there were 12 (0.1%) cervical stenoses, and 37 (3.13%) urethral stenoses. Five-hundred-and-fifty-two patients were followed for one year and more: the results were overall very good.(ABSTRACT TRUNCATED AT 250 WORDS)

Enzyme production by genetically engineered Streptomyces strains: influence of culture conditions.

Production of various extracellular enzymes (the beta-lactamases from Streptomyces albus G, Streptomyces cacaoi, Actinomadura R39, and the DD-carboxypeptidase from Streptomyces R61) by genetically engineered Streptomyces lividans TK24 in Lennox broth medium reached a maximum after 36 to 48 h. Subsequently, the enzyme activity drastically decreased probably due to an increased pH value and the production of an inactivator by Streptomyces lividans. Protease activity did not seem to play a major role. The increased pH and inactivator synthesis are related to amino acid catabolism and generally result in cellularlysis. The use of a medium where the catabolism of amino acids was made less likely by the presence of glucose and NH(4)Cl and by buffering at pH 7.4 considerably inproved the yield. Furthermore, the water activity of the medium seemed to be an important parameter for the production of extracellular proteins by genetically engineered Streptomyces. Better production was observed when the water activity was decreased to 0.96-0.98 by addition of sucrose.Under those conditions, the concentration of extracellular enzyme reached about 0.3 g (1 g in the best case)/L of culture supernantant.

The diversity of the catalytic properties of class A beta-lactamases.

The catalytic properties of four class A beta-lactamases were studied with 24 different substrates. They exhibit a wide range of variation. Similarly, the amino acid sequences are also quite different. However, no relationships were found between the sequence similarities and the substrate profiles. Lags and bursts were observed with various compounds containing a large sterically hindered side chain. As a group, the enzymes could be distinguished from the class C beta-lactamases on the basis of the kappa cat. values for several substrates, particularly oxacillin, cloxacillin and carbenicillin. Surprisingly, that distinction was impossible with the kappa cat./Km values, which represent the rates of acylation of the active-site serine residue by the beta-lactam. For several cephalosporin substrates (e.g. cefuroxime and cefotaxime) class A enzymes consistently exhibited higher kappa cat. values than class C enzymes, thus belying the usual distinction between 'penicillinases' and 'cephalosporinases'. The problem of the repartition of class A beta-lactamases into sub-classes is discussed.

Engineering and overexpression of periplasmic forms of the penicillin-binding protein 3 of Escherichia coli.

Replacement of the 36 and 56 N-terminal amino acid residues of the 588-amino-acid-residue membrane-bound penicillin-binding protein 3 (PBP3) of Escherichia coli by the OmpA signal peptide allows export of F37-V577 PBP3 and G57-V577 PBP3 respectively into the periplasm. The modified ftsI genes were placed under the control of the fused lpp promoter and lac promoter/operator; expression of the truncated PBP3s was optimized by varying the copy number of the recombinant plasmids and the amount of LacI repressor, and export was facilitated by increasing the SecB content of the producing strain. The periplasmic PBP3s (yield 8 mg/l of culture) were purified to 70% protein homogeneity. They require the presence of 0.25 M NaCl to remain soluble. Like the membrane-bound PBP3, they undergo processing by elimination of the C-terminal decapeptide I578-S588, they bind penicillin in a 1:1 molar ratio and they catalyse hydrolysis and aminolysis of acyclic thioesters that are analogues of penicillin. The membrane-anchor-free PBP3s have ragged N-termini. The G57-V577 PBP3, however, is less prone to proteolytic degradation than the F37-V577 PBP3.

Primary and predicted secondary structures of the Actinomadura R39 extracellular DD-peptidase, a penicillin-binding protein (PBP) related to the Escherichia coli PBP4.

As derived from gene cloning and sequencing, the 489-amino-acid DD-peptidase/penicillin-binding protein (PBP) produced by Actinomadura R39 has a primary structure very similar to that of the Escherichia coli PBP4 [Mottl, Terpstra & Keck (1991) FEMS Microbiol. Lett. 78, 213-220]. Hydrophobic-cluster analysis of the two proteins shows that, providing that a large 174-amino-acid stretch is excluded from the analysis, the bulk of the two polypeptide chains possesses homologues of the active-site motifs and secondary structures found in the class A beta-lactamase of Streptomyces albus G of known three-dimensional structure. The 174-amino-acid insert occurs at equivalent places in the two PBPs, between helices alpha 2 and alpha 3, away from the active site. Such an insert is unique among the penicilloyl serine transferases. It is proposed that the Actinomadura R39 PBP and E. coli PBP4 form a special class, class C, of low-Mr PBPs/DD-peptidases. A vector has been constructed and introduced by electrotransformation in the original Actinomadura R39 strain, allowing high-level expression and secretion of the DD-peptidase/PBP (250 mg.l-1). The gene encoding the desired protein is processed differently in Actinomadura R39 and Streptomyces lividans. Incorrect processing in Streptomyces lividans leads to a secreted protein which is inert in terms of DD-peptidase activity and penicillin-binding capacity.

Purification and properties of three endo-beta-1,4-xylanases produced by Streptomyces sp. strain S38 which differ in their ability to enhance the bleaching of kraft pulps*(2).

In the presence of xylan, Streptomyces sp. strain S38 secretes three xylanases (Xyl1, Xyl2, and Xyl3) that were purified to protein homogeneity and characterized. When used in bleach boosting tests on kraft hardwood and softwood, Xyl1, a family-11 enzyme, was more effective than Xyl2 and Xyl3 that belonged to family-10. Xyl1 was fully responsible for the biodelignification potential of the culture supernatants with a minimal effective amount of 10 IU per gram of dry pulp for both softwood and hardwood pulp. Complete conventional CEDED bleaching sequences showed that enzymatic pretreatment (20 IU/g dry pulp) could result in active chlorine savings of 8.6 and 4.9 kg/ton of dry pulp with hardwood and softwood, respectively. The purified enzymes were totally devoid of cellulase activity on CM-cellulose and their activities were optimal at about 60 degrees C and pH 6. Moreover, the V(max) value of Xyl1 at 50 degrees C measured on birchwood xylan (5,700 µmoles/min/mg prot.) was significantly higher than those of Xyl2 and Xyl3 whereas their K(m) values were similar. Their half-lives at 50 degrees C were larger than 16 h but sharply decreased at 60 degrees C where the family-11 Xyl1 was less stable (t(1/2)(60 degrees C) = 10 min) than both family-10 enzymes Xyl2 (t(1/2)(60 degrees C) = 30 min) and Xyl3 (t(1/2)(60 degrees C) = 70 min).