Effect of type of fiber, site of fermentation, and method of analysis on digestibility of soluble and insoluble fiber in rabbits

The effect of type of fiber, site of fermetation, method for quantifying insoluble and soluble dietary fiber, and their correction for intestinal mucin on fiber digestibility were examined in rabbits. Three diets differing in soluble fiber were formulated (8.5% soluble fiber, on DM basis, in the low soluble fiber [LSF] diet; 10.2% in the medium soluble fiber [MSF] diet; and 14.5% in the high soluble fiber [HSF] diet). They were obtained by replacing half of the dehydrated alfalfa in the MSF diet with a mixture of beet and apple pulp (HSF diet) or with a mix of oat hulls and soybean protein (LSF diet). Thirty rabbits with ileal T-cannulas were used to determine ileal and fecal digestibility. Cecal digestibility was determined by difference between fecal and ileal digestibility. Insoluble fiber was measured as NDF, insoluble dietary fiber (IDF), and in vitro insoluble fiber, whereas soluble fiber was calculated as the difference between total dietary fiber (TDF) and NDF (TDF_NDF), IDF (TDF-IDF), and in vitro insoluble fiber (TDF-in vitro insoluble fiber). The intestinal mucin content was used to correct the TDF and soluble fiber digestibility. Ileal and fecal concentration of mucin increased from the LSF to the HSF diet group (P < 0.01). Once corrected for intestinal mucin, ileal and fecal digestibility of TDF and soluble fiber increased whereas cecal digestibility decreased (P < 0.01). Ileal digestibility of TDF increased from the LSF to the HSF diet group (12.0 vs. 28.1%; P < 0.01), with no difference in the cecum (26.4%), resulting in a higher fecal digestibility from the LSF to the HSF diet group (P < 0.01). Ileal digestibility of insoluble fiber increased from the LSF to the HSF diet group (11.3 vs. 21.0%; P < 0.01), with no difference in the cecum (13.9%) and no effect of fiber method, resulting in a higher fecal digestibility for rabbits fed the HSF diet compared with the MSF and LSF diets groups (P < 0.01).Fecal digestibility of NDF was higher compared with IDF or in vitro insoluble fiber (P < 0.01). Ileal soluble fiber digestibility was higher for the HSF than for the LSF diet group (43.6 vs. 14.5%; P < 0.01) and fiber method did not affect it. Cecal soluble fiber digestibility decreased from the LSF to the HSF diet group (72.1 vs. 49.2%; P < 0.05). The lowest cecal and fecal soluble fiber digestibility was measured using TDF-NDF (P < 0.01). In conclusion, a correction for intestinal mucin is necessary for ileal TDF and soluble fiber digestibility whereas the selection of the fiber method has a minor relevance. The inclusion of sugar beet and apple pulp increased the amount of TDF fermented in the small intestine.

Application of the Boundary Method to the determination of the properties of the beam cross-sections

Using the 3-D equations of linear elasticity and the asylllptotic expansion methods in terms of powers of the beam cross-section area as small parameter different beam theories can be obtained, according to the last term kept in the expansion. If it is used only the first two terms of the asymptotic expansion the classical beam theories can be recovered without resort to any "a priori" additional hypotheses. Moreover, some small corrections and extensions of the classical beam theories can be found and also there exists the possibility to use the asymptotic general beam theory as a basis procedure for a straightforward derivation of the stiffness matrix and the equivalent nodal forces of the beam. In order to obtain the above results a set of functions and constants only dependent on the cross-section of the beam it has to be computed them as solutions of different 2-D laplacian boundary value problems over the beam cross section domain. In this paper two main numerical procedures to solve these boundary value pf'oblems have been discussed, namely the Boundary Element Method (BEM) and the Finite Element Method (FEM). Results for some regular and geometrically simple cross-sections are presented and compared with ones computed analytically. Extensions to other arbitrary cross-sections are illustrated.

Stabilization of microtubule dynamics by estramustine by binding to a novel site in tubulin: A possible mechanistic basis for its antitumor action

The cellular targets for estramustine, an antitumor drug used in the treatment of hormone-refractory prostate cancer, are believed to be the spindle microtubules responsible for chromosome separation at mitosis. Estramustine only weakly inhibits polymerization of purified tubulin into microtubules by binding to tubulin (Kd, ≈30 μM) at a site distinct from the colchicine or the vinblastine binding sites. However, by video microscopy, we find that estramustine strongly stabilizes growing and shortening dynamics at plus ends of bovine brain microtubules devoid of microtubule-associated proteins at concentrations substantially below those required to inhibit polymerization of the microtubules. Estramustine strongly reduced the rate and extent both of shortening and growing, increased the percentage of time the microtubules spent in an attenuated state, neither growing nor shortening detectably, and reduced the overall dynamicity of the microtubules. Significantly, the combined suppressive effects of vinblastine and estramustine on the rate and extent of shortening and dynamicity were additive. Thus, like the antimitotic mechanisms of action of the antitumor drugs vinblastine and taxol, the antimitotic mechanism of action of estramustine may be due to kinetic stabilization of spindle microtubule dynamics. The results may explain the mechanistic basis for the benefit derived from combined use of estramustine with vinblastine or taxol, two other drugs that target microtubules, in the treatment of hormone-refractory prostate cancer.

Global variation in the genetic and biochemical basis of diamondback moth resistance to Bacillus thuringiensis

Insecticidal proteins from the soil bacterium Bacillus thuringiensis (Bt) are becoming a cornerstone of ecologically sound pest management. However, if pests quickly adapt, the benefits of environmentally benign Bt toxins in sprays and genetically engineered crops will be short-lived. The diamondback moth (Plutella xylostella) is the first insect to evolve resistance to Bt in open-field populations. Here we report that populations from Hawaii and Pennsylvania share a genetic locus at which a recessive mutation associated with reduced toxin binding confers extremely high resistance to four Bt toxins. In contrast, resistance in a population from the Philippines shows multilocus control, a narrower spectrum, and for some Bt toxins, inheritance that is not recessive and not associated with reduced binding. The observed variation in the genetic and biochemical basis of resistance to Bt, which is unlike patterns documented for some synthetic insecticides, profoundly affects the choice of strategies for combating resistance.

Structural basis for efficient phosphorylation of 3′-azidothymidine monophosphate by Escherichia coli thymidylate kinase

The crystal structures of Escherichia coli thymidylate kinase (TmpK) in complex with P1-(5′-adenosyl)-P5-(5′-thymidyl)pentaphosphate and P1-(5′-adenosyl)P5-[5′-(3′-azido-3′-deoxythymidine)] pentaphosphate have been solved to 2.0-Å and 2.2-Å resolution, respectively. The overall structure of the bacterial TmpK is very similar to that of yeast TmpK. In contrast to the human and yeast TmpKs, which phosphorylate 3′-azido-3′-deoxythymidine 5′-monophosphate (AZT-MP) at a 200-fold reduced turnover number (kcat) in comparison to the physiological substrate dTMP, reduction of kcat is only 2-fold for the bacterial enzyme. The different kinetic properties toward AZT-MP between the eukaryotic TmpKs and E. coli TmpK can be rationalized by the different ways in which these enzymes stabilize the presumed transition state and the different manner in which a carboxylic acid side chain in the P loop interacts with the deoxyribose of the monophosphate. Yeast TmpK interacts with the 3′-hydroxyl of dTMP through Asp-14 of the P loop in a bidentate manner: binding of AZT-MP results in a shift of the P loop to accommodate the larger substituent. In E. coli TmpK, the corresponding residue is Glu-12, and it interacts in a side-on fashion with the 3′-hydroxyl of dTMP. This different mode of interaction between the P loop carboxylic acid with the 3′ substituent of the monophosphate deoxyribose allows the accommodation of an azido group in the case of the E. coli enzyme without significant P loop movement. In addition, although the yeast enzyme uses Arg-15 (a glycine in E. coli) to stabilize the transition state, E. coli seems to use Arg-153 from a region termed Lid instead. Thus, the binding of AZT-MP to the yeast TmpK results in the shift of a catalytic residue, which is not the case for the bacterial kinase.

Structural basis for heterogeneous kinetics: Reengineering the hairpin ribozyme

The RNA cleavage reaction catalyzed by the hairpin ribozyme shows biphasic kinetics, and chase experiments show that the slow phase of the reaction results from reversible substrate binding to an inactive conformational isomer. To investigate the structural basis for the heterogeneous kinetics, we have developed an enzymatic RNA modification method that selectively traps substrate bound to the inactive conformer and allows the two forms of the ribozyme-substrate complex to be separated and analyzed by using both physical and kinetic strategies. The inactive form of the complex was trapped by the addition of T4 RNA ligase to a cleavage reaction, resulting in covalent linkage of the 5′ end of the substrate to the 3′ end of the ribozyme and in selective and quantitative ablation of the slow kinetic phase of the reaction. This result indicates that the inactive form of the ribozyme-substrate complex can adopt a conformation in which helices 2 and 3 are coaxially stacked, whereas the active form does not have access to this conformation, because of a sharp bend at the helical junction that presumably is stabilized by inter-domain tertiary contacts required for catalytic activity. These results were used to improve the activity of the hairpin ribozyme by designing new interfaces between the two domains, one containing a non-nucleotidic orthobenzene linkage and the other replacing the two-way junction with a three-way junction. Each of these modified ribozymes preferentially adopts the active conformation and displays improved catalytic efficiency.

A method for directed evolution and functional cloning of enzymes

A general scheme is described for the in vitro evolution of protein catalysts in a biologically amplifiable system. Substrate is covalently and site specifically attached by a flexible tether to the pIII coat protein of a filamentous phage that also displays the catalyst. Intramolecular conversion of substrate to product provides a basis for selecting active catalysts from a library of mutants, either by release from or attachment to a solid support. This methodology has been developed with the enzyme staphylococcal nuclease as a model. An analysis of factors influencing the selection efficiency is presented, and it is shown that phage displaying staphylococcal nuclease can be enriched 100-fold in a single step from a library-like ensemble of phage displaying noncatalytic proteins. Additionally, this approach should allow one to functionally clone natural enzymes, based on their ability to catalyze specific reactions (e.g., glycosyl transfer, sequence-specific proteolysis or phosphorylation, polymerization, etc.) rather than their sequence- or structural homology to known enzymes.

Analysis of Respiratory Chain Regulation in Roots of Soybean Seedlings1

Changes in the respiratory rate and the contribution of the cytochrome (Cyt) c oxidase and alternative oxidase (COX and AOX, respectively) were investigated in soybean (Glycine max L. cv Stevens) root seedlings using the 18O-discrimination method. In 4-d-old roots respiration proceeded almost entirely via COX, but by d 17 more than 50% of the flux occurred via AOX. During this period the capacity of COX, the theoretical yield of ATP synthesis, and the root relative growth rate all decreased substantially. In extracts from whole roots of different ages, the ubiquinone pool was maintained at 50% to 60% reduction, whereas pyruvate content fluctuated without a consistent trend. In whole-root immunoblots, AOX protein was largely in the reduced, active form at 7 and 17 d but was partially oxidized at 4 d. In isolated mitochondria, Cyt pathway and succinate dehydrogenase capacities and COX I protein abundance decreased with root age, whereas both AOX capacity and protein abundance remained unchanged. The amount of mitochondrial protein on a dry-mass basis did not vary significantly with root age. It is concluded that decreases in whole-root respiration during growth of soybean seedlings can be largely explained by decreases in maximal rates of electron transport via COX. Flux via AOX is increased so that the ubiquinone pool is maintained in a moderately reduced state.

Dopamine transporters are markedly reduced in Lesch-Nyhan disease in vivo.

Dopamine (DA) deficiency has been implicated in Lesch-Nyhan disease (LND), a genetic disorder that is characterized by hyperuricemia, choreoathetosis, dystonia, and compulsive self-injury. To establish that DA deficiency is present in LND, the ligand WIN-35,428, which binds to DA transporters, was used to estimate the density of DA-containing neurons in the caudate and putamen of six patients with classic LND. Comparisons were made with 10 control subjects and 3 patients with Rett syndrome. Three methods were used to quantify the binding of the DA transporter so that its density could be estimated by a single dynamic positron emission tomography study. These approaches included the caudate- or putamen-to-cerebellum ratio of ligand at 80-90 min postinjection, kinetic analysis of the binding potential [Bmax/(Kd x Vd)] using the assumption of equal partition coefficients in the striatum and the cerebellum, and graphical analysis of the binding potential. Depending on the method of analysis, a 50-63% reduction of the binding to DA transporters in the caudate, and a 64-75% reduction in the putamen of the LND patients was observed compared to the normal control group. When LND patients were compared to Rett syndrome patients, similar reductions were found in the caudate (53-61%) and putamen (67-72%) in LND patients. Transporter binding in Rett syndrome patients was not significantly different from the normal controls. Finally, volumetric magnetic resonance imaging studies detected a 30% reduction in the caudate volume of LND patients. To ensure that a reduction in the caudate volume would not confound the results, a rigorous partial volume correction of the caudate time activity curve was performed. This correction resulted in an even greater decrease in the caudate-cerebellar ratio in LND patients when contrasted to controls. To our knowledge, these findings provide the first in vivo documentation of a dopaminergic reduction in LND and illustrate the role of positron emission tomography imaging in investigating neurodevelopmental disorders.

Reduced surface expression of transforming growth factor beta receptor type II in mitogen-activated T cells from Sézary patients.

Sézary syndrome (SzS), the leukemic form of cutaneous T-cell lymphoma, is characterized by clonal proliferation of CD4+ T cells and immune dysfunctions, raising the possibility of cytokine-related abnormalities. We previously described a decreased response to the growth-inhibitory effects of transforming growth factor type beta (TGF-beta) in SzS T cells accompanied by apparent loss of surface type II TGF-beta receptor (TGF beta RII). To specifically determine if defects exist in TGF beta RII protein expression and/or transport in SzS patients, we developed a sensitive flow cytometric method to detect TGF beta RII on the surface and intracellularly in the CD4+ T cells. Our results indicate that unlike normal CD4+ T cells, CD4+ T cells from 9 of 12 SzS patients expressed little, if any, surface TGF beta RII in response to mitogen stimulation. At the intracellular level, however, pools of TGF beta RII were comparable to those in normal CD4+ T cells. This indicates that defective trafficking of this inhibitory cytokine receptor may contribute significantly to the development of this disease.

Basis for selection of improved carbohydrate-binding single-chain antibodies from synthetic gene libraries.

A technique is described for the simultaneous and controlled random mutation of all three heavy or light chain complementarity-determining regions (CDRs) in a single-chain Fv specific for the O polysaccharide of Salmonella serogroup B. Sense oligonucleotides were synthesized such that the central bases encoding a CDR were randomized by equimolar spiking with A, G, C, and T at a level of 10% while the antisense strands contained inosine in the spiked regions. Phage display of libraries assembled from the spiked oligonucleotides by a synthetic ligase chain reaction demonstrated a bias for selection of mutants that formed dimers and higher oligomers. Kinetic analyses showed that oligomerization increased association rates in addition to slowing dissociation rates. In combination with some contribution from reduced steric clashes with residues in heavy-chain CDR2, oligomerization resulted in functional affinities that were much higher than that of the monomeric form of the wild-type single-chain Fv.

Method for the characterization of Fresnel lens flux transfer performance

Fresnel lenses and other faceted or micro-optic devices are increasingly used in multiple applications like solar light concentrators and illumination devices, just to name some representative. However, it seems to be a certain lack of adequate techniques for the assessment of the performance of final fabricated devices. As applications are more exigent this characterization is a must. We provide a technique to characterize the performance of Fresnel lenses, as light collection devices. The basis for the method is a configuration where a camera images the Fresnel lens aperture. The entrance pupil of the camera is situated at the focal spot or the conjugate of a simulated solar source. In this manner, detailed maps of the performance of different Fresnel lenses are obtained for different acceptance angles.

Geologia e petrologia da ilha de São Sebastião (Estado de São Paulo)

A ilha de São Sebastião consta principalmente de rochas alcalinas que formam um maciço de 300 km2 aproximadamente, constituindo o terceiro em área no Brasil. Apresenta-se em um "stock" alongado segundo NE-SW, encaixado em estruturas de gnais. As formações geológicas encontradas consistem em 1 - Granitos e Gnais (ARQUEANO), 2 - Eruptivas básicas (RÉTICO), 3 - Eruptivas alcalinas (JURÁSSICO) e 4 - Depósitos recentes (HOLOCENO). O método de estudo empregado foi o petrográfico e a coluna geológica estabelecida em base de dados petrográficos, tectônicos e fisiográficos. O arqueano é determinado por definição dos seus tipos petrográficos (1- gnais facoidal, 2- oligoclásio-gnais, 3- hornblenda-gnais, 4- biotita-gnais e 5- microlina-granito) idênticos aos concorrentes no considerado arqueano do Brasil meridional. O triássico (rético) é conferido às rochas básicas (diabásios e basaltos) pela sua semelhança tectônica e petrográfica com as congêneres que cortam de maneira semelhante o arqueano no continente. A "mise-en-place" das eruptivas alcalinas (1- Nordmarkito, 2- Biotita-pulaskito, 3- Pulaskito, 4- Nefelina-sienito, 5- Foiaito, 6- Essexito-foiaito, 7- Essexito e 8- Teralito) pode ser considerada jurássica devido suas relações com as eruptivas básicas referidas réticas, pois na praia do Bonete (foto 14) observa-se um dique de nordmarkito cortando outro de diabásio. As eruptivas quartzo-dioríticas (quartzo-microdiorito e quartzo-andesito) cortam as alcalinas no cume do Zabumba, indicando sua idade mais moderna que estas. Além deste fato, preenchem linhas de fraturas tectônicas recentes, como as falhas ao longo do canal de São Sebastião, indicando que a topografia deveria ser a mesma que a atual para permitir rios efusivos ao nível do canal ou que pelo menos toda a zona de extrusão estivesse, como hoje está, em superfície. Os depósitos aluviais marinhos e continentais são considerados recentes, (holocênicos) pelo favor da topografia onde se dispõe, ocupando o fundo os vales e os bordos do atual modelado costeiro, idade esta conferida em base fisiográfica. A tectônica que afetou a ilha de São Sebastião participa da que atuou em todo o litoral meridional brasileiro. Pode-se distinguir duas fases distintas: na primeira ocorreram as erupções básicas e as alcalinas subsidiárias e na segunda deram-se os falhamentos escalonados em blocos basculados para NW, com as fraturas de tensão preenchidas pelas eruptivas quartzo-dioríticas. Toda a atividade tectônica foi regulada pela direção NE-SW privilegiada da estrutura do arqueano, correspondente a antigos eixos dos dobramentos laurencianos e huronianos. A geomorfologia da ilha consta de uma antiga superfície de erosão rematada até a senilidade, - o peneplano cretáceo, hoje reduzida às cristas culminares do maciço alcalino e às satélites das estruturas gnáissicas, desnivelada pelo falhamento em blocos e ligeiramente adernada para NW devido ao basculamento. Ao lado desta topografia vestigial existe o modelado atual da ilha caracterizado por uma juventude do estágio evolutivo. Esta escultura foi inaugurada com os últimos levantamentos epirogênicos que ascenderam as eruptivas alcalinas plutônicas a mais de 1.300 m sobre o nível do mar. O modelado costeiro apresenta uma costa típica de submergência com esculturas em rias, no estágio da juventude. A presença de terraceamentos marinhos de abrasão, atualmente elevados cerca de 20 a 30 m, lembra as oscilações epirogênicas ou eustáticas do litoral.

Investigating the influence of surfactants on the stabilization of aqueous reduced graphene oxide dispersions and the characteristics of their composite films

The stabilization of reduced graphene oxide (RGO) sheets in aqueous dispersion using a wide range of surfactants of anionic, non-ionic and zwitterionic type has been investigated and compared under different conditions of pH, surfactant and RGO concentration, or sheet size. The observed differences in the performance of the surfactants were rationalized on the basis of their chemical structure (e.g., alkylic vs. aromatic hydrophobic tail or sulfonic vs. carboxylic polar head), thus providing a reference framework in the selection of appropriate surfactants for the processing of RGO suspensions towards particular purposes. RGO-surfactant composite paper-like films were also prepared through vacuum filtration of the corresponding mixed dispersions and their main characteristics were investigated. The composite paper-like films were also electrochemically characterized. Those prepared with two specific surfactants exhibited a high capacitance in relation to their surfactant-free counterpart.

A robust and fast method for 6DoF motion estimation from generalized 3D data

Nowadays, there is an increasing number of robotic applications that need to act in real three-dimensional (3D) scenarios. In this paper we present a new mobile robotics orientated 3D registration method that improves previous Iterative Closest Points based solutions both in speed and accuracy. As an initial step, we perform a low cost computational method to obtain descriptions for 3D scenes planar surfaces. Then, from these descriptions we apply a force system in order to compute accurately and efficiently a six degrees of freedom egomotion. We describe the basis of our approach and demonstrate its validity with several experiments using different kinds of 3D sensors and different 3D real environments.