The multigene families of actinoporins (part II): Strategies for heterologous production in Escherichia coli.

The sea anemone venom contains pore-forming proteins (PFP) named actinoporins, due to their purification from organisms belonging to Actiniaria order and its ability to form pores in sphingomyelin-containing membranes. Actinoporins are generally basic, monomeric and single-domain small proteins (∼20 kDa) that are classified as α-type PFP since the pore formation in membranes occur through α-helical elements. Different actinoporin isoforms have been isolated from most of the anemones species, as was analyzed in the first part of this review. Several actinoporin full-length genes have been identified from genomic-DNA libraries or messenger RNA. Since the actinoporins lack carbohydrates and disulfide bridges, their expression in bacterial systems is suitable. The actinoporins heterologous expression in Escherichia coli simplifies their production, replaces the natural source reducing the ecological damage in anemone populations, and allows the production of site-specific mutants for the study of the structure-function relationship. In this second part of the review, the strategies for heterologous production of actinoporins in Escherichia coli are analyzed, as well as the different approaches used for their purification. The activity of the recombinant proteins with respect to the wild-type is also reviewed.

The multigene families of actinoporins (part I): Isoforms and genetic structure.

Actinoporins are basic pore-forming proteins produced by sea anemones, with molecular weight around 20 kDa showing high affinity for sphingomyelin-containing membranes. Most sea anemones produce more than one actinoporin isoform differing in isoelectric point, molecular weigth and cytolytic activity. Examples of sea anemones with actinoporin isoforms are: Actinia equina with at least five isoform genes; Actinia tenebrosa, three isoforms; Actinia fragacea, five isoforms; Actineria villosa, Phyllodiscus semoni, Stichodactyla helianthus and Oulactis orientalis, with two isoforms each one, and Heteractis crispa with twenty-four isoforms. Additionally, thirty-four different amino acid sequences were deduced from fifty-two nucleotide sequences of Heteractis magnifica toxins suggesting the presence of a large number of isoforms or allelic variants. Many amino acidic changes in the isoforms are located in important regions for pore formation. The genetic structure of actinoporins comprises a pre-propeptide and a mature toxin region; therefore, actinoporins could be synthetized in the Golgi apparatus as precursor forms. The subsequent maturation of the toxins involves a proteolytic processing during secretion. Here we hypothesize that sea anemones could have suffered duplication, conversion and mutation of genes that produced multigene families as an efficient response to evolutionary pressure, leading to successful strategies of predatory and defensive function.

Interest of energy dispersive X-ray microanalysis to characterize the surface composition of milk powder particles.

Energy dispersive X-ray (EDX) is a technique rarely used for organic powders. Nevertheless, this technique is of great interest in the characterization of milk particle surface. In order to validate the method, the EDX technique was tested on pure milk components, on model powders composed of different ratio of lactose/whey proteins and on whole milk powders presenting or not free fat at the surface. For all these powders, satisfactory results were obtained with correct experimental atomic percentages in comparison with expected theoretical percentages. The technique was then applied to skimmed and whole milk powders sieved in 4 fractions. The surface and the core (cut particle) were analyzed by EDX and compared. A relationship between the particle size and the surface composition was observed. X-ray photoelectron spectroscopy (XPS) often used to characterize milk powder surface, however no differences were observed between surface and core composition using this method. The depth of analysis by EDX is far more significant (1 µm) in comparison to that of the XPS (5 nm); hence it was concluded that the analysis of cut particle by EDX was not interesting since too close to the results obtained at the surface. Finally, the technique was coupled with XPS and successful hypothesis concerning composition gradients were done.

Advances in food powder agglomeration engineering.

Food powders are used in everyday life in many ways and offer technological solutions to the problem of food production. The natural origin of food powders, diversity in their chemical composition, variability of the raw materials, heterogeneity of the native structures, and physicochemical reactivity under hydrothermal stresses contribute to the complexity in their behavior. Food powder agglomeration has recently been considered according to a multiscale approach, which is followed in the chapter layout: (i) at the particle scale, by a presentation of particle properties and surface reactivity in connection with the agglomeration mechanisms, (ii) at the mechanisms scale, by describing the structuration dynamics of agglomerates, (iii) at the process scale, by a presentation of agglomeration technologies and sensors and by studying the stress transmission mode in the powder bed, and finally (iv) by an integration of the acquired knowledge, thanks to a dimensional analysis carried out at each scale.

Comparative study of particle structure evolution during water sorption: skim and whole milk powders.

Surface composition of dairy powders influences significantly a quantity of functional properties such as rehydration, caking, agglomeration. Nevertheless, the kinetic of water uptake by the powders was never directly related to the structure and the composition of the surface. In this work, the effect of relative humidity on the structural reorganization of two types of dairy powder was studied. The water-powder interaction for industrial whole milk powder, and skim milk powder was studied using dynamic vapor sorption. The water sorption isotherms were fitted with a Brunner-Emmet-Teller model and each stage of the sorption curve was analyzed with a Fickian diffusion. The water content in the monolayer predicted for each powder and the moisture diffusivity calculated were discussed and compared. Concurrently, powders microstructure and powders surface under variable relative humidity were assessed by X-ray photoelectron spectroscopy, scanning electron microscopy coupled with energy dispersive X-ray and atomic force microscopy. A correlation between the data obtained from the sorption isotherms and the modifications of structure allowed us to conclude that powder microstructure and chemical state of the components could play an important role in determining the water diffusivity.

Adhesion and detachment kinetics of several strains of Staphylococcus aureus subsp. aureus under three different experimental conditions.

The kinetics of adhesion of five Staphylococcus aureus subsp. aureus strains (CECT 976, 4459, 4465, 4466 and 5191) to polypropylene at 25 degrees C in the absence of nutrients (PBS medium) were initially compared. Those strains with the highest (CECT 4459) and the lowest (CECT 976) adhesion levels were selected for further studying the effects of a nutrient-rich adhesion-promoting medium (TSB plus 1% glucose-TSBG) as well as of a conditioning film consisting of dried mussel cooking juices (MCJ) on adhesion to and detachment from polypropylene surfaces. Adhesion kinetics were properly described by an empirical model in the absence of conditioning film. The maximum adhesion level was much higher in the presence of TSBG than in PBS, decreasing sharply in both cases after 10-15 h. In contrast, adhesion increased exponentially during 25 h in the presence of dried MCJ. Clear differences were thus found in different media, and it suggests that cleaning strategies should vary under different conditions. The comparison of the adhesion strengths under the different experimental conditions showed that the persistence was highest when biofilms were formed on MCJ, which indicates that cells would remain longer as a source of cross-contamination. Some biofilms were examined by electronic microscopy, and different structures were observed under the different experimental conditions. It is concluded that the study of biofilm formation by S. aureus is necessary to establish efficient control systems in the food industry.

Primary chemotherapy with sequential docetaxel followed by docetaxel and epirubicin in large operable breast cancer.

UNLABELLED: Primary chemotherapy is increasingly used in patients with large operable breast cancer. Docetaxel and epirubicin are the most active agents in breast cancer treatment. PURPOSE: To evaluate clinical response rate, breast conserving surgery and pathological response rate in patients with large operable breast cancer treated with docetaxel followed by docetaxel and epirubicin as primary chemotherapy. PATIENTS AND METHODS: Patients with operable breast cancer more than 3 cm in the longest diameter with T2N0, T2N1 and T3N0 disease were enrolled. Patients were treated with three cycles of docetaxel 100 mg/m2 followed by three cycles of docetaxel 75 mg/m2 and epirubicin 90 mg/m2 prior to surgery. RESULTS: Sixty-five patients were enrolled between 09/2002 and 12/2005. The median age was 48.9 years and 72.3% were premenopausal. Median tumour size was 4.26 cm, 10.8% were T3 tumours and 38.5% had clinical positive lymph nodes. Of the tumours 58.5% were grade 1/2, 33.9% ER positive and 21.5% c-erb negative. All six cycles were administered to 62 patients; six cycles were delayed and five had dose reductions. Complete clinical response occurred in 41.5% of patients and partial response in 49.2%. Breast conserving surgery was performed in 30% of patients however it was feasible in 57%. Complete pathological response occurred in both primary tumour and nodes in 28%, and in 34% just in the primary tumour. Nine percent of cases had neutropenia and 7.7% febrile neutropenia, and two cases had a hypersensitivity reaction to docetaxel. One associated treatment death occurred. CONCLUSION: Docetaxel followed by epirubicin and docetaxel as primary chemotherapy results in a high clinical and pathological response rate. The majority of adverse events were predictable and manageable.

Comparison of pore-forming ability in membranes of a native and a recombinant variant of Sticholysin II from Stichodactyla helianthus.

Sticholysin II (St II) a potent cytolysin from the sea anemone Stichodactyla helianthus was obtained by recombinant procedures exhibiting six histidine residues in its N-terminus (St IIn6H). The functional comparison between St II and St IIn6H showed a lesser pore-forming ability for the recombinant than for the native in human or rat red blood cells (RBC) and in large unilamellar vesicles (LUV) of different phospholipid composition. However, binding of St IIn6H to small unilamellar vesicles (SUV) was higher with regard to St II. The explanation to the different permeabilizing capacity of both protein variants is not clear, but a different anchoring of St IIn6H to the lipid bilayer could delay the organization of the competent pore into membrane.

Effect of pH on the conformation, interaction with membranes and hemolytic activity of sticholysin II, a pore forming cytolysin from the sea anemone Stichodactyla helianthus.

Sticholysin II (St II) is a pore forming cytolysin obtained from the sea anemone Stichodactyla helianthus. Incubation of diluted St II solutions at different pHs (ranging from 2.0 to 12) slightly changes the secondary structure of the protein. These changes are particularly manifested at high pH. Similarly, the intrinsic fluorescence of the protein indicates a progressive opening of the protein structure when the pH increases from acidic (2.0) to basic (12). These modifications are only partially reversible and do not produce any significant increase in the small capacity of the protein to bind hydrophobic dyes (ANS or Prodan). Experiments carried out with model membranes show a reduced capacity of binding to egg phosphatidyl choline:sphingomyelin (1:1) liposomes both at low (2.3) and high (11.5) pH. Preincubation of the protein in the 2. 5-9.0 pH range does not modify its hemolytic activity, measured in human red blood cells at pH 7.4. On the other hand, preincubation at pH 11.5 drastically reduces the hemolytic activity of the toxin. This strong reduction takes place without measurable modification of the toxin ability to be adsorbed to the red blood cell surface. This indicates that preincubation at high pH irreversibly reduces the capacity of the toxin to form pores without a significant decrease in its binding capacity. The present results suggest that at pH > or = 10 St II experiences irreversible conformational changes that notably reduce its biological activity. This reduced biological activity is associated with a partial defolding of the protein, which seems to contradict what is expected in terms of a molten globule formalism.

Kinetics and mechanism of St I modification by peroxyl radicals.

St I is a toxin present in the Caribbean Sea anemone Stichodactyla helianthus which is highly hemolytic in the nanomolar concentration range. Exposure of the toxin to free radicals produced in the pyrolysis of 2,2'-azobis(2-amidinopropane) hydrochloride leads to a progressive loss of hemolytic activity. This loss of hemolytic activity is accompanied by extensive modification of tryptophan residues. On the average, three tryptophan residues are modified by each inactivated toxin. The loss of hemolytic activity of St I takes place without significant changes in the protein structure, as evidenced by the similarity of the fluorescence and CD spectra of native and modified proteins. Also, the native and modified ensembles present a similar resistance to their denaturation by guanidinium chloride. The hemolytic behavior and the performance of the toxin at the single-channel level when incorporated to black lipid membranes suggest that the modified ensemble can be considered as composed of inactive toxins and active toxins whose behavior is similar to that of the native proteins. These results, together with the lack of induction time in the activity loss, suggest that the fall of hemolytic activity takes place by an all-or-nothing inactivation mechanism in which the molecules become inactive when a critical amino acid residue is modified.

Phase and surface properties of lipid bilayers containing neoglycolipids.

The physical properties conferred to DPPC bilayers by including neoglycolipids composed by two different trisaccharides: mannose-mannose-mannose (3M) and glucose-mannose-glucose (GMG) attached to a cholesterol (cho) and a distearylglycerol (diC18) lipid moiety by a spacer were evaluated by means of the measurement of the electrokinetic potential and interfacial fluorescent probes. The phase properties measured with diphenylhexatriene (DPH) were correlated with the surface properties measured with merocyanine 540, dansyl, and Laurdan probes. The results show that the surface properties of large unilamellar vesicles depend on the sugar exposure to the water phase and also on the hydrocarbon moiety by which it is anchored to the bilayer. The combination of the cholesterol moiety with the saccharide attenuates the cooperativity decrease induced by the cholesterol moiety without the sugar portion. The neoglycolipid GMG-diC18 promotes opposite effects affecting slightly the cooperativity at the hydrocarbon core of DPPC and displacing the phase transition temperature to higher values. The presence of neoglycolipid with diC18 introduces defects in the packing at the interface of the membrane in the gel state. It is concluded that a relatively low proportion of neoglycolipids affects significantly the interfacial properties of DPPC bilayers in large unilamellar vesicles in the absence of changes at the membrane bulk at 25 degrees C.

Modification of sticholysin II hemolytic activity by free radicals.

Sticholysin II is a highly hemolytic toxin present in the caribbean sea anemone Stichodactyla helianthus. Pre-incubation of St II with 2,2'-azobis(2-amidinopropane), a source of peroxyl radicals in air saturated solution, readily reduces its hemolytic activity. Analysis of the amino acids present in the protein after its modification shows that only tryptophan groups are significantly modified by the free radicals. According to this, the loss of hemolytic activity correlates with the loss of the protein intrinsic fluorescence. The results indicate that, at high toxin concentrations, nearly a tryptophan residue and 0.2 toxin molecules are inactivated by each radical introduced into the system. Association of St II to multilamellar liposomes (egg yolk phosphatidyl choline:sphingomyelin 1:1) increases the toxin intrinsic fluorescence, indicating a more hydrophobic average environment of the five tryptophan groups of the protein. In agreement with this, incorporation of St II to the liposomes reduces the rate of fluorescence loss during its modification by free radicals, particularly at long incubation times. These results are explained in terms of two populations of tryptophans that are quenched at different rates by acrylamide and whose rates of inactivation by free radicals are also different.

Estudio multicentrico, abierto, no comparativo sobre la seguridad y eficacia de sulbactam cefoperazona en infecciones bacterianas moderadas y severas / Non comparative multicentric study over security and efficacy of sulbactam cefoperazone in moderate and severe in bacterials infections

La eficacia y seguridad de sulbactam/cefoperazona fue evaluada en un estudio multicéntrico, abierto, no comparativo, usado como monoterapia. Doscientos dieciseis pacientes entraron inicialmente al estudio, siendo excluidos 42, por haber recibido antibioticoterapia concomiante. Ciento setenta y cuatro pacientes adultos (99M, 75 F), con rango de edad de 13 a 83 años fueron tratados con sulbactam/cefoperazona como monoterapia, en proporción 1:2, vía endovenosa, presentando 102 (59 por ciento) pacientes infecciones severas, y, 72(41 por ciento) infecciones moderadas. La localización de las infecciones fueron: piel y tejidos blandos (n=54,31 por ciento), abdominales (n=54,31 por ciento), infecciones respiratorias bajas (n=40,23 por ciento), tracto biliar (n=9,5 por ciento), tracto urinario (n=9,5 por ciento), área ginecológica (n=3,2 por ciento) y otras (n=5,3 por ciento). Ciento sesenta y uno (93 por ciento) de las infecciones fueron agudas, nueve (5 por ciento) crónicas y, 4 (2 por ciento) recurrentes. Los gérmenes obtenidos con mayor frecuencia el cultivo inicial fueron P. aeruginosa (27,16 por ciento), E.coli (n=23,13 por ciento), E.cloacae (n=14,8 por ciento), S. aureus (n=7,4 por ciento). La dosis diaria administrada de sulbactam/cefoperazona osciló entre 3 y 9 gr/día (media 4,6g/día), durante 1-70 días (media 10 días). Ciento cuarenta y cuatro pacientes fueron evaluados clínicamente, obteniéndose curación en 112 pacientes (78 por ciento), mejoría en 24 (17 por ciento), y falla en 8 pacientes (5 por ciento). Se incluyeron 96 pacientes para evaluación bacteriológica, reportándose erradicación en 88 (91 por ciento) y, persistencia en 8 (9 por ciento). Se presentaron efectos adversos en 9 pacientes (5 por ciento): rash, prolongación del PTT, cefalea, escalofríos y diarrea. Con un 95 por ciento de respuesta clínica satisfactoria, 91 por ciento de erradicación bacteriológica y buena tolerancia concluimos que sulbactam/cefoperazona usado como monoterapia es una buena elección para el tratamiento de infecciones moderadas y severas.