Functional Diversity of Heat-labile Toxins (LT) Produced by Enterotoxigenic Escherichia coli

Heat-labile toxins (LTs) have ADP-ribosylation activity and induce the secretory diarrhea caused by enterotoxigenic Escherichia coli (ETEC) strains in different mammalian hosts. LTs also act as adjuvants following delivery via mucosal, parenteral, or transcutaneous routes. Previously we have shown that LT produced by human-derived ETEC strains encompass a group of 16 polymorphic variants, including the reference toxin (LT1 or hLT) produced by the H10407 strain and one variant that is found mainly among bacterial strains isolated from pigs (LT4 or pLT). Herein, we show that LT4 ( with six polymorphic sites in the A (K4R, K213E, and N238D) and B (S4T, A46E, and E102K) subunits) displays differential in vitro toxicity and in vivo adjuvant activities compared with LT1. One in vitro generated LT mutant (LTK4R), in which the lysine at position 4 of the A subunit was replaced by arginine, showed most of the LT4 features with an similar to 10-fold reduction of the cytotonic effects, ADP-ribosylation activity, and accumulation of intracellular cAMP in Y1 cells. Molecular dynamic studies of the A subunit showed that the K4R replacement reduces the N-terminal region flexibility and decreases the catalytic site crevice. Noticeably, LT4 showed a stronger Th1-biased adjuvant activity with regard to LT1, particularly concerning activation of cytotoxic CD8(+) T lymphocytes when delivered via the intranasal route. Our results further emphasize the relevance of LT polymorphism among human-derived ETEC strains that may impact both the pathogenicity of the bacterial strain and the use of these toxins as potential vaccine adjuvants.

Induction of neutralizing antibodies in mice immunized with an amino-terminal polypeptide of Streptococcus mutans P1 protein produced by a recombinant Bacillus subtilis strain

The oral pathogen Streptococcus mutans expresses a surface protein, P1, which interacts with the salivary pellicle on the tooth surface or with fluid-phase saliva, resulting in bacterial adhesion or aggregation, respectively. P1 is a target of protective immunity. Its N-terminal region has been associated with adhesion and aggregation functions and contains epitopes recognized by efficacious antibodies. In this study, we used Bacillus subtilis, a gram-positive expression host, to produce a recombinant N-terminal polypeptide of P1 (P1(39-512)) derived from the S. mutans strain UA159. Purified P1(39-512) reacted with an anti-full-length P1 antiserum as well as one raised against intact S. mutans cells, indicating preserved antigenicity. Immunization of mice with soluble and heat-denatured P1(39-512) induced antibodies that reacted specifically with native P1 on the surface of S. mutans cells. The anti-P1(39-512) antiserum was as effective at blocking saliva-mediated aggregation of S. mutans cells and better at blocking bacterial adhesion to saliva-coated plastic surfaces compared with the anti-full-length P1 antiserum. In addition, adsorption of the anti-P1 antiserum with P1(39-512) eliminated its ability to block the adhesion of S. mutans cells to abiotic surfaces. The present results indicate that P1(39-512), expressed and purified from a recombinant B. subtilis strain, maintains important immunological features of the native protein and represents an additional tool for the development of anticaries vaccines.

Functional and immunological characterization of a natural polymorphic variant of a heat-labile toxin (LT-I) produced by enterotoxigenic Escherichia coli (ETEC)

Heat-labile toxins (LT) encompass at least 16 natural polymorphic toxin variants expressed by wild-type enterotoxigenic Escherichia coli (ETEC) strains isolated from human beings, but only one specific form, produced by the reference ETEC H10407 strain (LT1), has been intensively studied either as a virulence-associated factor or as a mucosal/transcutaneous adjuvant. In the present study, we carried out a biological/immunological characterization of a natural LT variant (LT2) with four polymorphic sites at the A subunit (S190L, G196D, K213E, and S224T) and one at the B subunit (T75A). The results indicated that purified LT2, in comparison with LT1, displayed similar in vitro toxic activities (adenosine 3`,5`-cyclic monophosphate accumulation) on mammalian cells and in vivo immunogenicity following delivery via the oral route. Nonetheless, the LT2 variant showed increased adjuvant action to ovalbumin when delivered to mice via the transcutaneous route while antibodies raised in mice immunized with LT2 displayed enhanced affinity and neutralization activity to LT1 and LT2. Taken together, the results indicate that the two most frequent LT polymorphic forms expressed by wild ETEC strains share similar biological features, but differ with regard to their immunological properties.

Effects of Synthesis Conditions on the Nanostructure of Hybrid Sols Produced by the Hydrolytic Condensation of (3-Methacryloxypropyl)trimethoxysilane

Polysilsesquioxanes containing methacrylate pendant groups were prepared by the sol-gel process through hydrolysis and condensation of (3-methacryloxypropyl)trimethoxysilane (MPTS) dissolved in a methanol/methyl methacrylate (MMA) mixture. The effects of different water, MMA, and methanol contents, as well as of pH, on the nanoscopic and local structures of the system, at advanced stages of the condensation reaction, were studied by small-angle X-ray scattering (SAXS) and (29)Si nuclear magnetic resonance (NMR) spectroscopy, respectively. SAXS results indicate that the nanoscopic features of the hybrid sol could be described by a hierarchical model composed of two levels, namely (i) silsesquioxane (SSQO) nanoparticles Surrounded by the methacrylate pendant groups and the methanol/MMA mixture. and (ii) aggregation zones or islands containing correlated SSQO nanoparticles, embedded in the liquid medium. The (29)Si NMR results Show that the inner Structures of SSQO nanoparticles produced at pH 1 and 3 were built Up of polyhedral structures. mainly cagelike octamers and small linear oligomers, respectively. Irrespective of MMA and methanol contents, for a [H(2)O]/[MPTS] ratio higher than or equal to 1, the SSQO nailoparticles produced at pH I exhibit an average condensation degree (CD approximate to 69-87%) and average radius of gyration (R(g) approximate to 2.5 angstrom) larger than those produced at pH 3 (CD approximate to 48-67% and R(g) approximate to 1.5 angstrom). Methanol appears to act as a redispersion agent, by decreasing the number of particles inside the aggregation zones, while the addition of MMA induces a swelling of the aggregation zones.

Lipopeptides Produced by a Soil Bacillus Megaterium Strain

A soil microorganism identified as Bacillum megaterium was found to produce several antibiotics substances after growth for 20 h at 37A degrees C in a mineral culture medium. Analysis both by electron spray ionization (ESI) and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) identified these substances as lipopeptides. Predominant peaks at m/z 1,041 and m/z 1,065 revealed ions which are compatible with surfactins and lichenysins, respectively. Two other ions m/z 1,057 and m/z 1,464 were further studied by collision-induced dissociation (CID) unveiling an iturin A at the first and fengycins A and B at the second m/z peaks. The CID spectrum of the m/z 1,464 ion also suggests the existence of fengycins A and B variants in which Ile was changed to Val in the position 10 of the peptide moiety. Raw mixtures of all these compounds were also assayed for antibiotic features. The data enlighten the unusual diversity of the lipopeptide mixture produced by a sole Bacillus species.

Glucose-induced heat production, insulin secretion and lactate production in isolated Wistar rat pancreatic islets

Transplantation of pancreatic islets is efficient in improving the metabolic control and quality of life and in preventing severe hypoglycemia in patients with brittle type I diabetes mellitus. More accurate methods to assess islet viability would be extremely useful in designing target interventions for islet cytoprorection and in reducing the number of islets required to achieve insulin independence. Here we report on an application of calorimetry to evaluate the metabolic response of pancreatic islets to glucose stimulation. A significant increase in metabolic heat was produced by islet samples when consecutively subjected to 2.8 and 16.3 mmol L-1 glucose. Under these glucose concentrations, 1000 islets released average heat values of 9.16 +/- 0.71 mJ and 14.90 +/- 1.21 mJ over 50 min, respectively. Additionally, the glucose stimulation indexes were 1.67 +/- 0.30 for insulin. 1.72 +/- 0.13 for heat and 2.91 +/- 0.50 for lactate, raising the important possibility of substituting the secreted insulin index/ratio by the index/ratio of the heat released in the evaluation of Langerhans islets viability for transplantation. Altogether, Our results demonstrate the applicability of calorimetry to assess the quality of isolated pancreatic islets and to study vital islet functions. (c) 2008 Published by Elsevier B.V.

Stimulation of Acidic Reduction of Nitrite to Nitric Oxide by Soybean Phenolics: Possible Relevance to Gastrointestinal Host Defense

This study aimed to evaluate the potential of soybean-promoted acidic nitrite reduction and to correlate this activity with the content of phenolics and with the bactericidal activity against Escherichia coli O157:H7. Extracts of embrionary axes and cotyledons enriched in phenolics increased (center dot)NO formation at acidic pH at values that were 7.1 and 4.5 times higher, respectively, when compared to the reduction of the nonenriched extracts. Among the various phenolics accumulated in the soybean extracts, five stimulated nitrite reduction in the following decreasing order of potency: epicatechin gallate, chlorogenic acid, caffeic acid, galic acid and p-coumaric acid. Extracts of embrionary axes presented higher contents of epicatechin gallate and caffeic acid, compared to that of cotyledons, indicating a positive correlation between activity of the extracts and content of phenolics with regard to nitrite reducing activity. Soybean extracts enriched in phenolics interacted synergistically with acidified nitrite to prevent E. coli O157:H7 growth. The results suggest that soybean phenolics may interfere with the metabolism of (center dot)NO in an acidic environment by accelerating the reduction of nitrite, with a potential antimicrobial effect in the stomach.

Direct detection of underivatized chitooligosaccharides produced through chitinase action using capillary zone electrophoresis

Capillary electrophoresis with capacitively coupled contactless conductivity detection was successfully used to quantify N-acetylglucosamine and five N-acetyl-chitooligosaccharides (C2-C6) produced after reaction with a purified chitinase (TmChi) from Tenebrio molitor (Coleoptera). No derivatization process was necessary. The separation was developed using 10 mM NaOH with 10% (v/v) acetonitrile as background electrolyte and homemade equipment with a system that avoids the harmful effect of electrolysis. The limit of detection for all oligosaccharides was ca. 3 mu M, and the results indicated that the larger the oligosaccharide, the higher the sensitivity. Analysis of the chitooligosaccharides produced revealed that TmChi has an endolytic cleavage pattern with C5 as the best substrate (higher catalytic efficiency k(cat)/K-M) releasing C2 and C3. (c) 2007 Elsevier Inc. All rights reserved.

Expression, Purification, Bioactivity, and Partial Characterization of a Recombinant Human Bone Morphogenetic Protein-7 Produced in Human 293T Cells

Bone morphogenetic protein-7 (BMP-7) is a secreted multifunctional growth factor of the TGF-beta superfamily, which is predominantly known for its osteoinductive properties and emerging potential for treatment of kidney diseases. The mature 34-38 kDa disulfide-linked homodimer protein plays a key role in the differentiation of mesenchymal cells into bone and cartilage. In this study, the full-length sequence of hBMP-7 was amplified and, then, cloned, expressed, and purified from the conditioned medium of 293T cells stably transfected with a lentiviral vector. The mature protein dimer form was properly secreted and recognized by anti-BMP-7 antibodies, and the protein was shown to be glycosilated by treatment with exoglycosidase, followed by western blotting. Moreover, the activity of the purified protein was demonstrated both in vitro, by alkaline phosphatase activity in C2C12 cells, and in vivo by induction of ectopic bone formation in Balb/c Nude mice after 21 days, respectively. This recombinant protein platform may be very useful for expression of different human cytokines and other proteins for medical applications.

Wettability of Aqueous Rhamnolipids Solutions Produced by Pseudomonas aeruginosa LBI

The wetting behavior of rhamnolipids produced by Pseudomonas aeruginosa LBI strain grown on waste oil substrate and sodium dodecyl sulfate (SDS) on glass, polyethylene terephthalate (PET), poly(vinyl chloride) (PVC), poly(epsilon-caprolactone) (PCL) and polymer blend (PVC-PCL) was investigated by the measuring contact angle of sessile drops, to determine the wetting characteristics of rhamnolipids. The comparison of the wetting profiles showed that at low SDS and rhamnolipid concentrations, the contact angle increased and when the concentration of the surfactant increased further, the contact angle decreased. The blend surface (PVC-PCL) showed better wettability than the homopolymers themselves and the blend changed the surface hydrophobicity of the polymer, making it more hydrophilic. The rhamnolipids produced by the LBI strain exhibited superior wetting abilities than the chemical surfactant SDS one. This is the first work that evaluates the wetting properties of rhamnolipids on polymer blends.

Catalytic ethanolysis of soybean oil with immobilized lipase from Candida antarctica and (1)H NMR and GC quantification of the ethyl esters (biodiesel) produced

The catalytic ethanolysis of soybean oil with commercial immobilized lipase type B from Candida antarctica to yield ethyl esters (biodiesel) has been investigated. Transesterification was monitored with respect to the following parameters: quantity of biocatalyst, reaction time, amount of water added and turnover of lipase. The highest yields of biodiesel (87% by (1)H NMR; 82.9% by GC) were obtained after a reaction time of 24 h at 32 degrees C in the presence of lipase equivalent to 5.0% (w/w) of the amount of soybean oil present. The production of ethyl esters by enzymatic ethanolysis was not influenced by the addition of water up to 4.0% (v/v) of the alcohol indicating that it is possible to use hydrated ethanol in the production of biodiesel catalyzed by lipase. The immobilized enzyme showed high stability under moderate reaction conditions and retained its activity after five production cycles. The (1)H NMR methodology elaborated for the quantification of biodiesel in unpurified reaction mixtures showed good correlations between the signal areas of peaks associated with the alpha-methylene groups of the ethyl esters and those of the triacyl-glycerides in residual soybean oil. Monoacylglycerides, diacylglycerides and triglycerides could also be detected and quantified in the crude biodiesel using (1)H NMR spectroscopic and GC-FID chromatographic methods. The biodiesel production by enzymatic catalysis was promising. In this case, was produced a low concentration of glycerol (0.74%) and easily removed by water extraction. (C) 2010 Elsevier B.V. All rights reserved.

Structure and Applications of a Rhamnolipid Surfactant Produced in Soybean Oil Waste

Soybean oil soapstock was utilized as an alternative carbon source for the production of rhamnolipids by Pseudomonas aeruginosa LBI strain. The chemical composition and properties of the rhamnolipid mixture obtained were determined to define its potential applications. The chemical characterization of the rhamnolipid has revealed the presence of ten different homologues. The monorhamnolipid RhaC(10)C(10) and the dirhamnolipid Rha(2)C(10)C(10) were the main components of the mixture that showed predominance of 44% and 29%, respectively, after 144-h of cultivation. The biosurfactant was able to form stable emulsions with several hydrocarbons and showed excellent emulsification for soybean oil and chicken fat (100%). The rhamnolipid removed 67% of crude oil present in sand samples and presented antimicrobial activity against Bacillus cereus and Mucor miehei at 64 mu g/mL and inhibition of Neurospora crassa, Staphylococcus aureus, and Micrococcus luteus at 256 mu g/mL. The results demonstrated that the rhamnolipid produced in soybean oil soapstock can be useful in environmental and food industry applications.

Acupuncture for labour pain

Background: Acupuncture involves puncturing the skin with thin sterile needles at defined acupuncture points. Previous studies are inconclusive regarding the effect of acupuncture on labour pain, but some studies have found a reduction in the use of pharmacological pain relief when acupuncture is administered. The appropriate dose of acupuncture treatment required to elicit a potential effect on labour pain has not been fully explored. The dose is determined by many different factors, including the number of needles used and the intensity of the stimulation. In Sweden, manual stimulation of the needles is common practice when acupuncture is used for labour pain, but electrical stimulation of the needles, which gives a higher dose, could possibly be more effective. The overall aim of this thesis was to evaluate the effectiveness of acupuncture with manual stimulation (MA) of the needles as well as acupuncture with a combination of manual and electrical stimulation (EA) in reducing labour pain, compared with standard care without any form of acupuncture (SC). Methods: The study was designed as a three-armed randomised controlled trial in which 303 nulliparous women with normal pregnancies were randomised to MA, EA, or SC. The primary outcome was labour pain, assessed using the Visual Analogue Scale (VAS). Secondary outcomes were relaxation during labour, use of obstetric pain relief, and associations between maternal characteristics and labour pain and use of epidural analgesia respectively. Also, labour and infant outcomes, recollection of labour pain, and maternal experiences, such as birth experience and experience of the midwife, were investigated two months after the birth. The sample size calculation was based on the potential to discover a difference of 15 mm on the VAS. Data were collected during labour before the interventions, the day after birth, and two months later. Besides using the VAS, information was collected by means of study specific protocol, questionnaires and medical records. Results: The mean VAS scores were 66.4 in the MA group, 68.5 in the EA group, and 69.0 in the SC group (mean differences: MA vs. SC 2.6 95% CI -1.7 to 6.9, and EA vs. SC 0.6 95% CI -3.6 to 4.8). Other methods of pain relief were used less frequently in the EA group, including epidural analgesia, MA 61.4%, EA 46%, and SC 69.9%. (EA vs. SC OR 0.4 95% CI 0.2 to 0.7). No statistically significant differences were found in the recollection of labour pain between the three groups two months after birth (mean VAS score: MA 69.3, EA 68.7 and SC 70.1). A few maternal characteristics were associated with labour pain (age, dysmenorrhea, and cervix dilatation), but none of the investigated characteristics predicted the outcome of the acupuncture treatment in MA or EA. Women in the EA group experienced acupuncture as being effective for labour pain to a higher extent than women who received MA, MA 44.4%, EA 67.1% (EA vs. MA OR 2.4 95% CI 1.2 to 4.8). Women in the EA group also spent less time in labour (mean 500 min) than those who received MA (mean 619 min) and SC (mean 615 min) (EA vs. MA HR 1.4 95% CI 1.0 to1.9, EA vs. SC HR 1.4, 95% CI 1.1 to 2.0), and had less blood loss than women receiving SC, (EA vs. SC OR 0.1 95% CI 0.3 to 0.7). The women’s assessment of the midwife as being supportive during labour (MA 77.2%, EA 83.5%, SC 80%), overall satisfaction with midwife care (MA 100%, EA 97.5%, SC 98.7%), and having an overall positive childbirth experience (MA 64.6%, EA 61.0%, SC 54.3%) did not differ statistically. No serious side effects of the acupuncture treatment were reported. Conclusion: Acupuncture, regardless of type of stimulation, did not differ from standard care without acupuncture in terms of reducing women’s experience of pain during labour, or their memory of pain and childbirth overall two months after the birth. However, other forms of obstetric pain relief were less frequent in women receiving a combination of manual and electrical stimulation, suggesting that this method could facilitate coping with labour pain.

Guanylyl cyclase stimulation by homologous and heterologous natriuretic peptides in salmonids

A range of homologous (trout ANP, trout CNP, trout VNP) and heterologous (eel ANP, eel ANP-NH₂, rat ANP, porcine CNP) NPs were tested for their effect on guanylyl cyclase in gill and kidney membrane preparations from freshwater and seawater-acclimated rainbow trout and Atlantic salmon. All NPs stimulated guanylyl cyclase at 1 µmol l^-1in all preparations. ANP was the most potent stimulator of kidney guanylyl cyclase and CNP the most potent stimulator of guanylyl cyclase in gills. Some differences were apparent between the potencies of homologous and heterologous peptides at 1 µmol l^-1: tANP was more potent than rANP in the SW trout kidney and tCNP was more potent than pCNP in FW salmon tissues. While eANP was more potent than tANP in trout gills, it was less potent than tANP in FW salmon gills. However, there was no significant difference between the potencies of eANP and eANP-NH₂ in trout or salmon gills. Salinity did not affect guanylyl cyclase activity with the exception that trout ANP at 1 µmol l^-1was more potent in SW trout kidneys than in FW trout kidneys. These results suggest a predomination of NPR-A in the kidney and NPR-B in the gill. It appears that salmonid NPR-A and NPR-B are relatively promiscuous in their ligand affinity, with few differences in the potencies of trout and mammalian NPs and only small differences in cGMP production where these differences do occur.