Revisiting Polymer-Particle Interaction in PEO Solutions.

We have measured the electrophoretic mobility and diffusion coefficient of carboxylate-modified and sulfate-modified polystyrene latex particles in poly(ethylene oxide) aqueous solutions. Carboxylate-modified polystyrene particles have shown a bound polymeric layer as the surface net charge vanishes even at very low poly(ethylene oxide) concentration. The polymeric layer causes a lower electrophoretic mobility and slower Brownian diffusion than that corresponding to the bare particles. We show that the diffusion is the result of a significantly increased effective particle size 2rheff = 30 nm. This bound layer is not present in sulfate-modified polystyrene latex particles. The interaction between the carboxylate-modified particle surface and the macromolecules has been confirmed by means of atomistic computer simulations. The grafted acrylate copolymers, which come from the preparation procedure of the latex particles, confer more hydrophobic surface ready to interact with the polymer. The simulations suggest that the interaction is modulated not only by the nature of the acrylic acid monomer but also by the length of the grafted copolymer. Our results have important implications for particle selection in microrheology experiments.

Molecular and hydrodynamic properties of human epidermal growth factor receptor HER2 extracellular domain and its homodimer: Experiments and multi-scale simulations.

BACKGROUND: In a broad range of human carcinomas gene amplification leads to HER2 overexpression, which has been proposed to cause spontaneous dimerization and activation in the absence of ligand. This makes HER2 attractive as a therapeutic target. However, the HER2 homodimerization mechanism remains unexplored. It has been suggested that the "back-to-back" homodimer does not form in solution. Notwithstanding, very recently the crystal structure of the HER2 extracellular domain homodimer formed with a "back-to-head" interaction has been resolved. We intend to explore the existence of such interactions. METHODS: A combination of experiments, molecular dynamics and hydrodynamic modeling were used to monitor the transport properties of HER2 in solution. RESULTS & CONCLUSIONS: We have detected the HER2 extracellular domain homodimer in solution. The results show a high degree of molecular flexibility, which ultimately leads to quite higher values of the intrinsic viscosity and lower values of diffusion coefficient than those corresponding to globular proteins. This flexibility obeys to the open conformation of the receptor and to the large fluctuations of the different domains. We also report that for obtaining the correct hydrodynamic constants from the modeling one must consider the glycosylation of the systems. GENERAL SIGNIFICANCE: Conformational features of epidermal growth factor receptors regulate their hydrodynamic properties and control their activity. It is essential to understand the dynamics of these systems and the role of the specific domains involved. To find biophysical correlations between dynamics and macroscopic transport properties is of general interest for researches working in this area. This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O'Donoghue.

Willingness to pay and conjoint analysis to determine women's preferences for ovarian stimulating hormones in the treatment of infertility in Spain.

BACKGROUND: Despite many advances in assisted reproductive techniques (ART), little is known about preferences for technological developments of women undergoing fertility treatments. The aims of this study were to investigate the preferences of infertile women undergoing ART for controlled ovarian stimulation (COS) treatments; to determine the utility values ascribed to different attributes of COS treatments; and to estimate women's willingness to pay (WTP) for COS. METHODS: A representative sample of ambulatory patients ready to receive, or receiving, COS therapies for infertility were recruited from seven specialized private centres in six autonomous communities in Spain. Descriptive, inferential and conjoint analyses (CA) were used to elicit preferences and WTP. Attributes and levels of COS treatments were identified by literature review and two focus groups with experts and patients. WTP valuations were derived by a combination of double-bounded (closed-ended) and open questions and contingent ranking methods. RESULTS: In total, 160 patients [mean (standard deviation; SD) age: 35.8 (4.2) years] were interviewed. Over half of the participants (55.0%) had a high level of education (university degree), most (78.8%) were married and half (50.0%) had an estimated net income of >€1502 per month and had paid a mean (SD) €1194.17 (€778.29) for their most recent hormonal treatment. The most frequent causes of infertility were related to sperm abnormalities (50.3%). In 30.6% of cases, there were two causes of infertility. The maximum WTP for COS treatment was €800 (median) per cycle; 35.5% were willing to pay an additional €101-€300 for a 1-2% effectiveness gain in the treatment. Utility values (CA) showed that effectiveness was the most valued attribute (39.82), followed by costs (18.74), safety (17.75) and information sharing with physicians (14.93). CONCLUSIONS: WTP for COS therapies exceeds current cost. Additional WTP exists for 1-2% effectiveness improvement. Effectiveness and costs were the most important determinants of preferences, followed by safety and information sharing with physicians.

GnRH antagonist ganirelix prevents premature luteinization in IUI cycles: rationale for its use.

This study evaluated the efficacy of a gonadotrophin-releasing hormone antagonist (GnRHa) to prevent premature luteinization (PL) and examined its impact on intrauterine insemination (IUI) cycle outcome. A total of 662 patients who were undergoing IUI were evaluated. Ovarian stimulation was started on day 3 with recombinant (r)FSH, followed by the GnRHa and recombinant human chorionic gonadotrophin (rHCG). The overall incidence of PL was 11.5%. In patients with and without PL, the pregnancy rates (PR) were 22.4 (17/76) and 17.7% (104/586) respectively. Patients with PL were divided into two groups: (i) those with increased serum progesterone [PR was 10.7% (3/28) in this group]; and (ii) patients with elevated serum LH but normal progesterone concentrations [PR was 29.2% (14/48) in this group]. Patients in the first group who did not become pregnant (n = 22) were administered GnRHa in a second IUI cycle, and the PR was 18.1%; however, some patients (n = 6; 27.3%) in this group still had high serum progesterone concentrations. In the second group, patients (n = 26) with elevated serum LH in the previous cycle were administered GnRHa in another IUI and the PR was 23.1%. Use of a GnRHa in patients with PL who have had a previous unsuccessful IUI may be an alternative in future attempts.

El coasting es eficaz en la prevención del síndrome de hiperestimulación ovárica y no afecta a los resultados de la fecundación in vitro / Coasting is effective in preventing ovarian hyperstimulation syndrome without affecting the results of in vitro fertilization

Objetivo: Describir los resultados de los ciclos de FIV/ICSI en pacientes sometidas a coasting por hiperrespuesta a la estimulación ovárica controlada (EOC), así como la frecuencia de presentación y severidad del síndrome de hiperestimulación ovárica (SHO). Además, evaluar si la duración del coasting compromete los resultados del ciclo. Pacientes y métodos: Estudio retrospectivo de 42 ciclos de fecundación in vitro de pacientes con hiperrespuesta a la EOC sometidas a coasting (mantener la administración del análogo de la GnRH mientras se suprime la administración de gonadotropinas) hasta que los valores séricos de estradiol (E2) disminuyan a un valor "seguro" para administrar la hCG y programar la punción ovárica. Resultados: El valor de E2 sérico promedio al iniciar y finalizar el coasting fue de 6.427,6 ñ 227,4 pg/ml y de 2.454,7 ñ 155,8 pg/ml, respectivamente.Las tasas de implantación y de gestación fueron del 26,9 y del 53,8 por ciento respectivamente. No hubo SHO severo y sólo un 14,2 por ciento de SHO moderado. En aquellas pacientes en las que el coasting duró > 4 días, la tasa de gestación fue significativamente menor que en las que el coasting duró 4 días (28,6 frente a 57,9 por ciento; p < 0,05). La aparición de SHO fue igual en ambos grupos. Conclusiones: El coasting reduce la incidencia y severidad del SHO, evitando la cancelación del ciclo en pacientes de muy alto riesgo de desarrollar un SHO, lo que permite mantener excelentes tasas de embarazo y de implantación.Los resultados son mejores si el coasting no se prolonga más de 4 días. (AU)

Role of the ruvB gene in homologous and homeologous recombination in Rhizobium etli.

The Rhizobium etli ruvA and ruvB genes were cloned through a PCR-based approach, using degenerate primers matching conserved sectors in the amino acid sequences of RuvB from eight bacterial species. Comparative analysis of the predicted polypeptides for RuvA and RuvB of R. etli showed highly conserved blocks with the corresponding homologs in other bacteria; RuvB depicts characteristic motifs for DNA helicases (ATP-binding and DEXH-box motifs). An R. etli ruvB::loxP Sp mutant was constructed by interposon mutagenesis. This mutant was highly sensitive to DNA-damaging agents, such as methyl methanesulfonate and nitrofurantoin, implying a deficiency in DNA repair. Homologous and homeologous conjugational recombination was reduced almost tenfold in the ruvB::loxP Sp mutant; a recombination defect was also observed in assays employing recombination between small plasmids, albeit at a smaller magnitude. Although the ruvA and ruvB genes are contiguous in R. etli, complementation studies suggest that they are expressed independently.

Repeated sequences in bacterial chromosomes and plasmids: a glimpse from sequenced genomes.

To gain insight into the extent of exact DNA repeats in sequenced bacterial genomes and their plasmids, we analyzed the collection of completely sequenced bacterial genomes available at GenBank using the program Miropeats. This program draws graphical representations of exact DNA repeats in whole genomes. In this work, we present maps showing the extent and type (inverted or direct) of exact DNA repeats longer than 300 bp for the whole collection. These repeats may participate in a variety of events relevant for bacterial genome plasticity, such as amplifications, deletions, inversions, and translocations (via homologous recombination), as well as transposition. Additionally, we review recent data showing that high-frequency architectural variations in genomic structure occur at both the interspecies and interstrain levels.

Evaluation of the role of recA protein in plant virulence with recA mutants of Xanthomonas campestris pv. campestris.

Xanthomonas campestris pv. campestris NRRL B1459 recA mutants were isolated by recombination with an interrupted Rhizobium etli recA gene and selection of double recombinants. The mutants were impaired in homologous genetic recombination and in DNA repair as judged by their sensitivity to methyl-methane-sulfonate and to UV irradiation; these defects are complemented in trans by the R. etli recA gene. Virulence of X. campestris pv. campestris NRRL B1459 to cabbage is considerably diminished by the recA mutation. The recA mutation is not correlated with the frequency of occurrence of a genetic rearrangement that affects chemotaxis, plant virulence, and xanthan gum production.

Genetic analysis of the transcriptional arrangement of Azotobacter vinelandii alginate biosynthetic genes: identification of two independent promoters.

The study of alginate biosynthesis, the exopolysaccharide produced by Azotobacter vinelandii and Pseudomonas aeruginosa, might lead to different biotechnological applications. Here we report the cloning of A. vinelandii algA, the gene coding for the bifunctional enzyme phosphomannose isomerase-guanosine diphospho-o-mannose pyrophosphorylase (PMI-GMP). This gene was selected by the complementation for xanthan gum production of Xanthomonas campestris pv. campestris xanB-mutants, which lack this enzymatic activity. The complementing cosmid clones selected, besides containing algA, presented a gene coding for an alginate lyase activity (algL), and some of them also contained algD which codes for GDP-mannose dehydrogenase. We present here the characterization of the A. vinelandii chromosomal region comprising algD and its promoter region, algA and algL, showing that, as previously reported for P. aeruginosa, A. vinelandii has a cluster of the biosynthetic alginate genes. We provide evidence for the presence of an algD-independent promoter in this region which transcribes at least algL and algA, and which is regulated in a manner that differs from that of the algD promoter.

Control of AlgU, a member of the sigma E-like family of stress sigma factors, by the negative regulators MucA and MucB and Pseudomonas aeruginosa conversion to mucoidy in cystic fibrosis.

The alternative sigma factor AlgU (Pseudomonas aeruginosa sigma E) is required for full resistance of P. aeruginosa to oxidative stress and extreme temperatures. AlgU also controls conversion of P. aeruginosa to the mucoid, alginate-overproducing phenotype associated with lethal infections in cystic fibrosis patients. Mutations that cause conversion to mucoidy in cystic fibrosis isolates occur frequently in mucA, the second gene within the algU mucABCD gene cluster. Here we analyze the biochemical basis of conversion to mucoidy. MucA was shown to act as an anti-sigma factor by binding to AlgU and inhibiting its activity. MucB, another negative regulator of AlgU, was localized in the periplasm. MucB exerts its function from this compartment, since deletion of the leader peptide and the cytoplasmic location of MucB abrogated its ability to inhibit mucoidy. These data support a model in which a multicomponent system, encompassing an anti-delta factor and elements in the periplasmic compartment, modulates activity of AlgU. Since factors controlling AlgU are conserved in other gram-negative bacteria, the processes controlling conversion to mucoidy in P. aeruginosa may be applicable to the regulation of AlgU (sigma E) equivalents in other organisms.

Characterization of the genes coding for the putative sigma factor AlgU and its regulators MucA, MucB, MucC, and MucD in Azotobacter vinelandii and evaluation of their roles in alginate biosynthesis.

The study of the biosynthesis of alginate, the exopolysaccharide produced by Azotobacter vinelandii and Pseudomonas aeruginosa, has biotechnological and medical significance. We report here the identification of the A. vinelandii genes coding for the putative sigma factor AlgU and its negative regulators MucA and MucB through the suppression of the highly mucoid phenotype of an A. vinelandii strain by a plasmid encoding MucA and MucB. The sequences of the A. vinelandii algU, mucA, and mucB genes are highly homologous to those of the corresponding P. aeruginosa genes, AlgU shows 93% identity, and MucA and MucB are 64.4 and 63.9% identical, respectively. Forming part of the same operon as algU, mucA, and mucB, two additional genes (mucC and mucD) were identified and sequenced; the product of the former gene is homologous to ORF4 of Photobacterium sp. strain SS9, and that of the latter gene belongs to the HtrA serine protease family. Interestingly, the nonmucoid A. vinelandii UW136 had a 0.9-kb insertion within the algU gene. A strong correlation between AlgU activity and alginate production by A. vinelandii was also found, as reflected in the level of algD transcription.

Characterization of the gene coding for GDP-mannose dehydrogenase (algD) from Azotobacter vinelandii.

Azotobacter vinelandii presents a differentiation process leading to the formation of desiccation-resistant cysts. Alginate, the exopolysaccharide produced by this bacterium, has been postulated to have a role in cyst formation. Here, we report the cloning and characterization of the A. vinelandii gene coding for the enzyme GDP-mannose dehydrogenase (algD), which is the key enzyme for alginate synthesis in Pseudomonas aeruginosa. This gene has a high degree of similarity with the algD gene from P. aeruginosa, and similar proteins seem to be involved in algD regulation in both bacteria. We show the existence of two mRNA start sites; one of these sites corresponds to a promoter transcribed by RNA polymerase containing a sigma E subunit. An A. vinelandii algD mutant which is completely impaired in alginate production and which is unable to form desiccation-resistant cells was constructed. The effects of NH4, NO3, and NaCl concentrations on algD transcription for three A. vinelandii strains producing different alginate levels were evaluated. We found a strict correlation between alginate production and algD transcription for the three strains studied; however, the effects on algD transcription under the conditions studied were different for each strain. The nitrogen source regulates algD expression in the wild-type strain.

Two distinct loci affecting conversion to mucoidy in Pseudomonas aeruginosa in cystic fibrosis encode homologs of the serine protease HtrA.

Conversion to a mucoid, exopolysaccharide alginate-overproducing phenotype in Pseudomonas aeruginosa is associated with chronic respiratory infections in cystic fibrosis. Mucoidy is caused by muc mutations that derepress the alternative sigma factor AlgU, which in turn activates alginate biosynthetic and ancillary regulatory genes. Here we report the molecular characterization of two newly identified genes, algW and mucD, that affect expression of mucoidy. The algW gene, mapping at 69 min, was isolated on the basis of its ability to suppress mucoidy and reduce transcription of the alginate biosynthetic gene algD. The predicted primary structure of AlgW displayed similarity to HtrA (DegP), a serine protease involved in proteolysis of abnormal proteins and required for resistance to oxidative and heat stress in enteric bacteria. Inactivation of algW on the chromosome of the wild-type nonmucoid strain PAO1 caused increased sensitivity to heat, H2O2, and paraquat, a redox cycling compound inducing intracellular levels of superoxide. This mutation also permitted significant induction of alginate production in the presence of subinhibitory concentrations of paraquat. Two new genes, mucC and mucD, were identified immediately downstream of the previously characterized portion (algU mucA mucB) of the gene cluster at 67.5 min encoding the alternative sigma factor AlgU and its regulators. Interestingly, the predicted gene product of mucD also showed similarities to HtrA. Inactivation of mucD on the PAO1 chromosome resulted in conversion to the mucoid phenotype. The mutation in mucD also caused increased sensitivity to H2O2 and heat killing. However, in contrast to algW mutants, no increase in susceptibility to paraquat was observed in mucD mutants. These findings indicate that algW and mucD play partially overlapping but distinct roles in P. aeruginosa resistance to reactive oxygen intermediates and heat. In addition, since mutations in mucD and algW cause conversion to mucoidy or lower the threshold for its induction by reactive oxygen intermediates, these factors may repress alginate synthesis either directly by acting on AlgU or its regulators or indirectly by removing physiological signals that may activate this stress response system.

Biochemical characterization and posttranslational modification of AlgU, a regulator of stress response in Pseudomonas aeruginosa.

AlgU is homologous to the extreme heat shock sigma factor sigma E from enteric bacteria. In this work, AlgU was overproduced and purified and its function investigated at the biochemical level. AlgU was shown to associate with RNA polymerase and direct transcription of a target promoter. AlgU also exhibited multiple isoforms detected by 2D gel analysis. Treatment with a Ser/Thr phosphatase shifted the distribution of isoforms towards the basic side on 2D gels, suggesting that posttranslational modifications of AlgU may involve phosphorylation. The underphosphorylated forms of AlgU copurified with RNA polymerase. It is possible that phosphorylation affects AlgU activity or its stability.

Acute toxicological effects in rats treated with a mixture of commercially formulated products containing methyl parathion and permethrin.

The study examined the interactions between a commercial formulation of methyl parathion (CF-MP) and a commercially formulated product of permethrin (CF-PMT) in male rats. The acute toxicity (LD50 values) and brain cholinesterase activity were investigated as toxicological endpoints. Results indicated that CF-MP modified the acute toxicity of CF-PMT. When animals were treated with a mixture, the addition of 380 mg/kg of CF-MP reduced the LD50 of CF-PMT by only 9.0%; however, when rats received CF-MP at 464 mg/kg, the LD50 of CF-PMT was reduced by 37% (P < 0.001). Also, CF-PMT decreased the CF-MP-induced inhibition of cholinesterase activity by 50% (P < 0.05). It was interesting to observe that xylene, which is the most abundant component in the vehicle of both formulations, had no effect on the CF-MP-induced inhibition of the cholinesterase activity. There was no relation between lethality and the inhibition of the brain cholinesterase activity in rats treated with mixtures containing CF-MP+CF-PMT or with either commercially formulated product alone. Considering the increased toxicity observed in rats treated with CF-PMT+CF-MP, it would be advisable to investigate further the interactions between both pesticides.

Discrete amplifiable regions (amplicons) in the symbiotic plasmid of Rhizobium etli CFN42.

Frequent tandem amplification of defined regions of the genome, called amplicons, is a common characteristic in the genomes of some Rhizobium species, such as Rhizobium etli. In order to map these zones in a model Rhizobium replicon, we undertook an analysis of the plasticity patterns fostered by amplicons in the pSym (390 kb) of R. etli CFN42. Data presented in this article indicate the presence of four amplicons in pSym, used for the generation of tandem amplifications and deletions. The amplicons are large, ranging from 90 to 175 kb, and they are overlapping. Each amplicon is usually flanked by specific reiterated sequences. Formation of amplifications and deletions requires an active recA gene. All the amplicons detected are concentrated in a zone of roughly one-third of pSym, covering most of the symbiotic genes detected in this plasmid. No amplicons were detected in the remaining two-thirds of pSym. These data support the idea that most of the known symbiotic genes in this plasmid are located in a genomic region that is prone to the formation of frequent tandem amplification.

Genetic stability and xanthan gum production in Xanthomonas campestris pv. campestris NRRL B1459.

A transposon (Tn5-SC) was constructed that can be used to quantify genetic deletions or amplifications. This transposon was used to evaluate the genomic stability of Xanthomonas campestris pv. campestris NRRL B1459 and we found that the genome of this bacterium is as stable as other Gram-negative bacteria or even more stable. Homologous recombination between plasmid sequences was determined in strain NRRL B1459 and was found to occur at a similar level to that reported for other Gram-negative bacteria. We report here that in X.c.c. NRRL B1459 there is no straightforward correlation between the occurrence of genetic rearrangements and frequency of homologous recombination. These data are discussed with respect to the reported instability of strain NRRL B1459 for xanthan gum production.

Molecular cloning and characterization of the recA gene of Rhizobium phaseoli and construction of recA mutants.

The Rhizobium phaseoli recA gene has been cloned by interspecific complementation of the Fec phenotype of bacteriophage lambda. The cloned gene restored the recombination proficiency and conferred resistance to DNA-damaging agents (methyl methanesulfonate and nitrofurantoin) to an Escherichia coli recA mutant. The direction of transcription and the localization of the recA gene were determined by mutagenesis with phage MudIIPR13 and heterologous hybridization with an E. coli recA probe. An R. phaseoli recA::Spcr mutation was introduced in two R. phaseoli strains by homogenotization. The R. phaseoli recA mutants were more sensitive to DNA-damaging agents and exhibited a 100-fold reduction in recombination frequency as compared with their parental strains. A deletion of the symbiotic plasmid abolishing nodulation was found at high frequency (10(-2)) in R. phaseoli CNF42. This event was recA dependent. In R. phaseoli CFN285, two events of symbiotic instability were found at high frequency (10(-3]: one was a deletion in the symbiotic plasmid, and the other was the loss of whole symbiotic plasmid. In the CFN285 recA::Spcr mutant, only the loss of the symbiotic plasmid was observed.

Amplification and deletion of a nod-nif region in the symbiotic plasmid of Rhizobium phaseoli.

One remarkable characteristic of the genomes of some Rhizobium species is the frequent occurrence of rearrangements. In some instances these rearrangements alter the symbiotic properties of the strains. However, no detailed molecular mechanisms have been proposed for the generation of these rearrangements. To understand the mechanisms involved in the formation of rearrangements in the genome of Rhizobium phaseoli, we have designed a system which allows the positive selection for amplification and deletion events. We have applied this system to investigate the stability of the symbiotic plasmid of R. phaseoli. High-frequency amplification events were detected which increase the copy number of a 120-kb region carrying nodulation and nitrogen fixation genes two to eight times. Deletion events that affect the same region were also found, albeit at a lower frequency. Both kinds of rearrangements are generated by recombination between reiterated nitrogenase (nifHDK) operons flanking the 120-kb region.

Molecular rearrangements between two plasmids of the FII incompatibility group in different recombination--deficient Escherichia coli strains.

The frequencies and types of plasmid molecular rearrangements generated in different recombinant mutants which carried two plasmids of the FII incompatibility group were studied. The wild-type cells generated molecular rearrangements mainly by interplasmidic recombination with a frequency of 2.4 x 10(-6) per cell per cell doubling. Cells in which RecF was the principal recombination pathway generated different types of molecular rearrangements that involved either both plasmids or one of the plasmids and the chromosome. The frequencies of molecular rearrangements for these cells were 50-fold greater than those of wild-type cells. The recA- cells, even when the RecE pathway was derepressed, generated rearrangements only between one of the plasmids and the chromosome, at very low frequencies (10(-9]. In wild-type cells and in RecF cells, interplasmidic recombination generated mainly cointegrates carrying DNA deletions. These cointegrates were stable in recA- or recA- RecE+ cells, but unstable in wild-type or RecF+ cells. In the latter, the cointegrates generated smaller plasmids with different molecular structures at relatively low frequencies.