Microrheology of semiflexible filament solutions based on relaxation simulations.

We present an efficient computational methodology to obtain the viscoelastic response of dilute solutions of semiflexible filaments. By considering an approach based on the fluctuation-dissipation theorem, we were able to evaluate the dynamical properties of probe particles immersed in solutions of semiflexible filaments from relaxation simulations with a relatively low computational cost and higher precision in comparison to those based on stochastic dynamics. We used a microrheological approach to obtain the complex shear modulus and the complex viscosity of the solution through its compliance which was obtained directly from the dynamical properties of a probe particle attached to an effective medium described by a mesoscopic model, i.e., an effective filament model (EFM). The relaxation simulations were applied to assess the effects of the bending energy on the viscoelasticity of the semiflexible filament solutions, and our methodology was validated by comparing the numerical results to the experimental data on DNA and collagen solutions.

Dodecyltrimethylammonium bromide surfactant effects on DNA: Unraveling the competition between electrostatic and hydrophobic interactions.

We present a new study on the interaction of the DNA molecule with the surfactant dodecyltrimethylammonium bromide (DTAB), performed mainly with optical tweezers. Single-molecule force spectroscopy experiments performed in the low-force entropic regime allowed a robust characterization of the DNA-DTAB interaction, unveiling how the surfactant changes the mechanical properties of the biopolymer, the binding parameters, and the competition of the two mechanisms involved in the interaction: electrostatic attraction between the cationic surfactant heads and the negative phosphate backbone of the DNA and hydrophobic interactions between the tails of the bound DTAB molecules, which can result in DNA compaction in solution depending on the quantity of bound surfactant. Finally, force clamp experiments with magnetic tweezers and gel electrophoresis assays confirm that DTAB compacts DNA depending not only on the surfactant concentration but also on the conformation of the biopolymer in solution. The present study provides new insights on general aspects of the DNA-surfactant complexes formation, contributing to the fundamental knowledge of the physics of such interactions.

Size-dependent bandgap and particle size distribution of colloidal semiconductor nanocrystals.

A new analytical expression for the size-dependent bandgap of colloidal semiconductor nanocrystals is proposed within the framework of the finite-depth square-well effective mass approximation in order to provide a quantitative description of the quantum confinement effect. This allows one to convert optical spectroscopic data (photoluminescence spectrum and absorbance edge) into accurate estimates for the particle size distributions of colloidal systems even if the traditional effective mass model is expected to fail, which occurs typically for very small particles belonging to the so-called strong confinement limit. By applying the reported theoretical methodologies to CdTe nanocrystals synthesized through wet chemical routes, size distributions are inferred and compared directly to those obtained from atomic force microscopy and transmission electron microscopy. This analysis can be used as a complementary tool for the characterization of nanocrystal samples of many other systems such as the II-VI and III-V semiconductor materials.

ß-Cyclodextrin polymer binding to DNA: Modulating the physicochemical parameters.

Cyclodextrins and cyclodextrins-modified molecules have interesting and appealing properties due to their capacity to host components that are normally insoluble or poorly soluble in water. In this work, we investigate the interaction of a ß-cyclodextrin polymer (poly-ß-CD) with λ-DNA. The polymers are obtained by the reaction of ß-CD with epichlorohydrin in alkaline conditions. We have used optical tweezers to characterize the changes of the mechanical properties of DNA molecules by increasing the concentration of poly-ß-CD in the sample. The physical chemistry of the interaction is then deduced from these measurements by using a recently developed quenched-disorder statistical model. It is shown that the contour length of the DNA does not change in the whole range of poly-ß-CD concentration (<300µM). On the other hand, significant alterations were observed in the persistence length that identifies two binding modes corresponding to the clustering of ∼2.6 and ∼14 polymer molecules along the DNA double helix, depending on the polymer concentration. Comparing these results with the ones obtained for monomeric ß-CD, it was observed that the concentration of CD that alters the DNA persistence length is considerably smaller when in the polymeric form. Also, the binding constant of the polymer-DNA interaction is three orders of magnitude higher than the one found for native (monomeric) ß-CD. These results show that the polymerization of the ß-CD strongly increases its binding affinity to the DNA molecule. This property can be wisely used to modulate the binding of cyclodextrins to the DNA double helix.

Atividade biológica in vitro de própolis e óleos essenciais sobre o fungo Colletotrichum musae isolado de bananeira (Musa spp. ) / In vitro biological activity of propolis and essential oils on the fungus Colletotrichum musae isolated from banana Musa spp

RESUMO: No Brasil existem várias doenças fúngicas que acometem a bananeira. Destas, pode-se citar a antracnose, responsável por grandes prejuízos à cultura, cujo agente causal é o fungo Colletotrichum musae. A principal forma de controle dessa enfermidade é através da aplicação de fungicidas a base de tiabendazol ou tiofanato metílico. Esse manejo, embora eficiente, favorece o desenvolvimento de resistência do patógeno, causa danos ao ambiente e ao produtor, deixando ainda resíduos nos frutos. Esses fatores têm favorecido a busca por substâncias alternativas com capacidade de controlar o fungo e que não sejam nocivas ao ambiente e, principalmente, que sejam seguras ao consumidor final. Dentre as opções, surge o interesse pelo uso de certos óleos essenciais e da própolis, ambos conhecidos por possuírem propriedades fungicidas. O presente trabalho foi desenvolvido com o objetivo de determinar o potencial fungitóxico "in vitro" da própolis e dos óleos essenciais de palmarosa (Cymbopogon martinii), de teatree (Melaleuca alternifolia), de cravo (Eugenia caryophyllata), e de eucalipto (Corymbia citriodora), sobre Colletotrichum musae. O desenvolvimento experimental consistiu em adicionar inóculos fúngicos de 5 mm, obtidos a partir de colônias puras, ao meio de cultura BDA (batata-dextrose-ágar) acrescido das referidas substâncias em diferentes concentrações (0, 25, 50, 75, 100 e 125 µL/L). Paralelo aos tratamentos realizou-se teste com o fungicida padrão para comparações das médias. A eficiência das substâncias sobre o fungo foi determinada através das avaliações do crescimento micelial das colônias (média de duas medidas diametralmente opostas). Os valores de crescimento micelial obtidos foram utilizados também para o cálculo do índice de velocidade de crescimento micelial. O delineamento experimental utilizado foi inteiramente casualizado em esquema fatorial 5 x 6 + 1, (cinco substâncias em seis concentrações + fungicida), com cinco repetições. Os óleos de tea tree, cravo e palmarosa foram eficientes no controle do fungo Colletotrichum musae não diferindo do fungicida a partir da dose de 50 µL/L em todas as avaliações, apresentando potencial para controle em cultivos orgânicos ou em sistemas de manejo integrado.

ABSTRACT: In Brazil there are several fungi that cause diseases on banana plants. These include the "anthracnose", which is responsible for major crop losses and whose causative agent is the fungus Colletotrichum musae. The main way to control this disease is through the application of fungicides based on thiabendazole and thiophanate-methyl. Although this management is effective, it favors the development of pathogen resistance, which causes damage to the environment and producer and also leaves residues in fruits. These factors have encouraged the search for alternative substances to control the fungus and that are not harmful to the environment and particularly to the final consumer. Among the options, there is interest in the use of essential oils and propolis, both known to have antifungal activity. The present work was developed with the objective of determining the potential of propolis and essential oils of palmarosa (Cymbopogon martinii), tea tree (Melaleuca alternifolia), clove (Eugenia caryophyllata) and eucalyptus (Corymbia citriodora) in the in vitro control of the fungus Colletotrichum musae. The experimental development consisted in adding 5 mm fungal inoculants, obtained from pure colonies, in PDA culture (potato-dextrose-agar) plus the aforementioned substances in different concentrations (0, 25, 50, 75, 100 and 125 µL/L). At the same time as these treatments, we carried out a test with the fungicide to compare the averages. The efficiency of the substances on the fungus was determined through evaluations of the mycelial growth of the colonies (average of two diametrically opposed measures). The values of mycelial growth obtained were also used for the calculation of the speed index of the mycelial growth. The experimental design was completely randomized in 5 x 6 + 1 (5 substances in 6 concentrations + fungicide) factorial design, with 5 repetitions. The tea tree, clove and palmarosa oils were efficient in the control of the fungus Colletotrichum musae, which can be used as a control option in organic crops or in integrated management systems.

Dynamic scaling of polymer gels comprising nanoparticles.

We present dynamic light scattering (DLS) measurements of soft poly(methyl-methacrylate) (PMMA) and polyacrylamide (PA) polymer gels prepared with trapped bodies (latex spheres or magnetic nanoparticles). We show that the anomalous diffusivity of the trapped particles can be analyzed in terms of a fractal Gaussian network gel model for the entire time range probed by DLS technique. This model is a generalization of the Rouse model for linear chains extended for structures with power law network connectivity scaling, which includes both percolating and uniform bulk gel limits. For a dilute dispersion of strongly scattering particles trapped in a gel, the scattered electric field correlation function at small wavevector ideally probes self-diffusion of gel portions imprisoning the particles. Our results show that the time-dependent diffusion coefficients calculated from the correlation functions change from a free diffusion regime at short times to an anomalous subdiffusive regime at long times (increasingly arrested displacement). The characteristic time of transition between these regimes depends on scattering vector as approximately q(-2), while the time decay power exponent tends to the value expected for a bulk network at small q. The diffusion curves for all scattering vectors and all samples were scaled to a single master curve.

Identification of a receptor-binding region within domain 4 of the protective antigen component of anthrax toxin.

Anthrax toxin from Bacillus anthracis is a three-component toxin consisting of lethal factor (LF), edema factor (EF), and protective antigen (PA). LF and EF are the catalytic components of the toxin, whereas PA is the receptor-binding component. To identify residues of PA that are involved in interaction with the cellular receptor, two solvent-exposed loops of domain 4 of PA (amino acids [aa] 679 to 693 and 704 to 723) were mutagenized, and the altered proteins purified and tested for toxicity in the presence of LF. In addition to the intended substitutions, novel mutations were introduced by errors that occurred during PCR. Substitutions within the large loop (aa 704 to 723) had no effect on PA activity. A mutated protein, LST-35, with three substitutions in the small loop (aa 679 to 693), bound weakly to the receptor and was nontoxic. A mutated protein, LST-8, with changes in three separate regions did not bind to receptor and was nontoxic. Toxicity was greatly decreased by truncation of the C-terminal 3 to 5 aa, but not by their substitution with nonnative residues or the extension of the terminus with nonnative sequences. Comparison of the 28 mutant proteins described here showed that the large loop (aa 704 to 722) is not involved in receptor binding, whereas residues in and near the small loop (aa 679 to 693) play an important role in receptor interaction. Other regions of domain 4, in particular residues at the extreme C terminus, appear to play a role in stabilizing a conformation needed for receptor-binding activity.

Internalization of a Bacillus anthracis protective antigen-c-Myc fusion protein mediated by cell surface anti-c-Myc antibodies.

BACKGROUND: Anthrax toxin, secreted by Bacillus anthracis, consists of protective antigen (PA) and either lethal factor (LF) or edema factor (EF). PA, the receptor-binding component of the toxin, translocates LF or EF into the cytosol, where the latter proteins exert their toxic effects. We hypothesized that anthrax toxin fusion proteins could be used to kill virus-infected cells and tumor cells, if PA could be redirected to unique receptors found only on these cells. MATERIALS AND METHODS: To test this hypothesis in a model system, amino acids 410-419 of the human p62(c-myc) epitope were fused to the C-terminus of PA to redirect PA to the c-Myc-specific hybridoma cell line 9E10. RESULTS: The PA-c-Myc fusion protein killed both mouse macrophages and 9E10 hybridoma cells when administered with LF or an LF fusion protein (FP59), respectively. Similar results were obtained with PA, which suggests that PA-c-Myc used the endogenous PA receptor to enter the cells. By blocking the endogenous PA receptors on 9E10 cells with the competitive inhibitor PA SNKEDeltaFF, the PA-c-Myc was directed to an alternate receptor, i.e., the anti-c-Myc antibodies presented on the cell surface. The c-Myc IgG were proven to act as receptors because the addition of a synthetic peptide containing the c-Myc epitope along with PA SNKEDeltaFF further reduced the toxicity of PA-c-Myc + FP59. CONCLUSION: This study shows that PA can be redirected to alternate receptors by adding novel epitopes to the C-terminus of PA, enabling the creation of cell-directed toxins for therapeutic purposes.

[Helicobacter pylori, nonsteroidal anti-inflammatory agents and gastroduodenal changes]. / Helicobacter pylori, anti-inflamatórios não esteroides e alterações gastro-duodenais.

The author discusses the possible interactions between non-steroidal anti-inflammatory drugs (NSAIDs) and Helicobacter pylori (Hp) which may play an important role in the unleashing of gastroduodenal lesions. To our knowledge, AINEs have no influence on the prevalence of infection by Hp and the latter does not seem to influence the development and intensity of the lesions caused by NSAIDs.

Synthesis and expression of a gene coding for Erythrina trypsin inhibitor (ETI).

A gene coding for Erythrina trypsin inhibitor (ETI) was designed, based on the published N-terminal sequence of the protein, and synthesized by an oligonucleotide-directed single strand break-repair mechanism. Direct expression from the expression vector pBtac1 was unsuccessful. A construct, encoding an extended methionyl N-terminal amino acid was expressed from the vector pET12a which supplies a signal sequence for export to the periplasm. Most of the expressed protein was located in the cytoplasm but because the periplasm is an environment conducive to the formation of disulphide bridges, only periplasmic protein was extracted. Cyanogen bromide cleavage at the sole methionyl residue removed the undesired amino acid residues that remained after signal sequence peptidase processing. The resultant ETI was assayed against trypsin and tissue plasminogen activator and found to have activity similar to that of natural ETI.

Site-directed mutagenesis of the synthetic Erythrina trypsin/tissue plasminogen activator (tPA) inhibitor encoding-gene to compare the interaction of Erythrina and soybean trypsin inhibitor with tPA.

Erythrina trypsin inhibitor (ETI) has good structural and sequence homology with soybean trypsin inhibitor (STI). However, STI does not inhibit tPA. From the three-dimensional structure of ETI it was known that the N-terminus of the molecule forms a finger-like structure stabilized by hydrogen bonds and hydrophobic interactions. In addition, the N-terminal finger region is located in close proximity to the reactive site loop and the N-terminal residue (Val) is bound up in the finger region. In STI the N-terminal region is located in close proximity to the reactive site loop and is folded into a structure similar to that in ETI. It was hypothesized that the N-terminal region is stabilized as in ETI and that the N-terminal residue of STI (Asp), because of its hydrophilic nature, is not involved in the structured N-terminal finger region of this protein. This leaves Asp1 of STI free to form an ion pair with Lys60 of trypsin, when STI and trypsin interact. When amino acid sequences of trypsin and the C-terminus of tPA are aligned for optimum homology, it is seen that there are a number of insertion sequences in tPA that are thought to be accommodated in the form of protrusions. One of these can be seen to occur in the region that lies opposite the Lys60 region of trypsin. It is suggested in this work that the N-terminal Asp of STI and this protrusion of tPA sterically prevent the two proteins from approaching close enough for binding and inhibition to occur. A modified form of ETI was produced with an Asp residue N-terminal to Val to simulate the N-terminal region of STI. The active sites were titrated against trypsin and assayed against tPA. The results showed that the modified form of ETI had activity towards tPA similar to that of STI. This evidence indicates strongly that the N-terminal Asp of STI prevents its binding to and inhibiting tPA.

Investigation into the nature of a Bacillus promoter cloned into a promoter-probe plasmid.

The alpha-amylase-coding gene (amy) of Bacillus amyloliquefaciens NCP1 was cloned into the Bacillus subtilis promoter probe vector pPL603b.1, using a BglII digest of chromosomal DNA. The resulting plasmid, pVC102, was shown to have a BglII site within the insert. It was determined that this was the result of the fortuitous co-cloning of 2.88-kb and 0.92-kb BglII fragments separated in NCP1 DNA by approx. 3 kb. Unexpectedly, this co-cloning was readily repeated. Subcloning showed that while the 2.88-kb amy-bearing fragment was sufficient for amylase production, it might not have been capable of promoting sufficient levels of chloramphenicol resistance under the conditions used in the cloning experiments. The promoter on the 0.92-kb BglII fragment was more efficient, although its sequence differed from the canonical promoter sequence recognised by B. subtilis RNA polymerase E.sigma 43. As other promoter-bearing fragments from NCP1 DNA operated equally efficiently when cloned into pPL603b.1, the reason for the repeated co-cloning of the 2.88-kb and 0.92-kb NCPI BglII fragments may well be due to structural parameters, whereby certain nucleotide sequences are more readily cloned than others.

Observations on the gastric mucosa of rheumatic patients before and after ibuprofen administration as studied by the pentagastrin test, endoscopy, and light and electron microscopy.

The initial results of a study set up to investigate the gastric mucosa in rheumatic patients receiving ibuprofen are described. The study involved seven male patients, aged between 17 to 70 years, suffering from various rheumatic diseases. All patients received a daily dose of 1200 mg of ibuprofen per os divided into three fractions and taken over periods of treatment ranging from one to six weeks. On the data obtained by the gastric secretion test, endoscopy, and specific histological and ultrastructure studies it is concluded that, in the cases investigated, ibuprofen could not be shown to be responsible for any significant modification of the gastric mucosa.