Resonant behaviour of the barrier of YBa2Cu3O7 grain boundary Josephson junctions fabricated on bicrystalline substrates with different geometries

We have analyzed a resonant behavior in the dielectric constant associated to the barrier of YBa2Cu3O7 (YBCO) grain boundary Josephson junctions (GBJJs) fabricated on a wide variety of bicrystalline substrates: 12° [0 0 1] tilt asymmetric, 24° [0 0 1] tilt asymmetric, 24° [0 0 1] tilt symmetric, 24° [1 0 0] tilt asymmetric, 45° [1 0 0] tilt asymmetric and 24° [0 0 1] tilt symmetric +45° [1 0 0] tilt asymmetric bicrystals. The resonance analysis allows us to estimate a more appropriate value of the relative dielectric constant, and so a more adequate value for the length L of the normal N region assuming a SNINS model for the barrier. In this work, the L dependence on the critical current density Jc has been investigated. This analysis makes possible a single representation for all the substrate geometries independently on around which axes the rotation is produced to generate the grain boundary. On the other hand, no clear evidences exist on the origin of the resonance. The resonance frequency is in the order of 1011 Hz, pointing to a phonon dynamic influence on the resonance mechanism. Besides, its position is affected by the oxygen content of the barrier: a shift at low frequencies is observed when the misorientation angle increases.

Effect of type of dietary carbohydrates on digestion, intestinal barrier and growth performance in 25-d weaned rabbits

Los objetivos globales de esta tesis han sido estudiar el efecto que los carbohidratos de la dieta ejercen sobre los rendimientos productivos, la barrera intestinal, y la digestión de animales destetados a 25 días de edad. Además se ha estudiado cuál es el mejor periodo para determinar la digestibilidad fecal tras el destete a esta edad. En el primer experimento se estudió el efecto de la fibra neutro detergente soluble (FNDS) sobre la barrera intestinal, digestión, microbiota intestinal y rendimientos productivos de gazapos en gazapos en la fase post-destete. Se diseñaron tres piensos isonutritivos en los que la única fuente de variación fueron los niveles de fibra soluble. A partir de una dieta control (AH) con 103 g/kg de materia seca de FNDS y alfalfa como fuente principal de fibra, se sustituyó la mitad de esta alfalfa por una mezcla de pulpa de remolacha y pulpa de manzana (75:25) en el pienso B-AP y por una mezcla de cascarilla y concentrado de proteína de soja (88:12) en el pienso OH, obteniéndose 131 y 79 g/kg de FNDS sobre materia seca, respectivamente. Los conejos se destetaron a 25 días y fueron alimentados con los piensos experimentales hasta los 35 días de edad, momento en el que se sacrificaron para la determinación de la digestibilidad ileal aparente (DIA) de la materia seca (MS), proteína bruta (PB) y almidón, la morfología de la mucosa, y actividad enzimática en el yeyuno, el tejido linfoide asociado a la mucosa, así como la microbiota intestinal. Para la determinación de la morfología de la mucosa se utilizaron adicionalmente 19 animales lactantes de 35 días de edad. Para el estudio de la tasa de mortalidad, se utilizaron 118 animales más por tratamiento que recibieron los piensos experimentales durante las dos semanas post-destete y posteriormente un pienso comercial hasta los 60 días de edad. Los animales recibieron durante todo el experimento medicación en el agua de bebida (100 ppm de apramicina sulfato y 120 ppm de tilosina tartrato). El nivel de fibra soluble mejoró los parámetros que se utilizaron para la caracterización del estado de la barrera intestinal. Los conejos alimentados con el mayor nivel de FNDS en el pienso presentaron una mayor longitud de los villi (P=0.001), un mayor ratio longitud villi/profundidad de las criptas (8.14; P=0.001), una mayor actividad disacaridásica (8671 μmol de glucosa/g de proteína; P=0.019), así como una mayor digestibilidad ileal (96.8%; P=0.002), observándose una reducción en el flujo ileal de almidón a medida que se incrementó el nivel de fibra soluble en el pienso (1,2 vs 0,5 g/d; P=0.001). Los animales lactantes a 35 días de edad presentaron un ratio longitud de villi/profundidad de las criptas menor que el observado en aquéllos alimentados con el pienso B-AP (6.70), pero superior al de los piensos AH y OH. Niveles inferiores de NDFS tendieron (P=0.074) a incrementar la respuesta inmune de tipo celular (linfocitos CD8+). El pienso también afectó a la producción de IL2 (CD25+; P=0.029; CD5+CD25+; P=0.057), pero sin llegar a establecerse una clara relación con el nivel de fibra soluble. La diversidad de la microbiota intestinal no se vio afectada por el pienso (P ≥ 0.38). Los animales alimentados con las piensos B-AP y AH presentaron una reducción en la frecuencia de detección de Clostridium perfringens tanto en íleon (P=0.062) como en ciego (4.3 vs. 17.6%, P =0.047), comparado con el pienso OH. Además la tasa de mortalidad (118 gazapos/pienso) disminuyó de 14.4% en el pienso OH a 5.1% en el pienso B-AP. Entre los 32 y los 35 días de edad se determinó la digestibilidad fecal aparente (14/pienso) de la materia seca (MS), energía bruta (EB), proteína bruta (PB), fibra neutro detergente (FND), fibra ácido detergente (FAD) y almidón. Este grupo, junto con otros nueve animales por tratamiento se utilizaron para determinar el peso del estómago y el ciego, la concentración cecal de ácidos grasos volátiles (AGV) y amoniaco (NH3), así como las tasas de similitud de la microbiota intestinal. Además se estudiaron los rendimientos productivos (35 animales/tratamiento) de los gazapos durante todo el período de cebo, consumiendo los piensos experimentales desde el destete hasta los 35 días y posteriormente un pienso comercial hasta los 60 días de edad. Niveles crecientes de FNDS mejoraron la digestibilidad fecal de la materia seca (MS) y energía (P<0.001). La inclusión FNDS aumentó de manera lineal el peso del contenido cecal (P=0.001) y el peso del aparato digestivo completo (P=0.008), y en los días previos al sacrificio disminuyó de manera lineal el consumo medio diario (P=0.040). Se observó además, una disminución lineal (P≤0.041) del pH del estómago. No se encontró relación entre el pH, la concentración y proporciones molares de AGV y el nivel de FNDS. El pienso pareció tener un efecto, incluso superior al de la madre, sobre la tasa de similitud de la microbiota, y los efectos fueron mayores a nivel cecal que ileal. La eficacia alimenticia aumentó de manera lineal en un 12% entre piensos extremos tras el destete (25- 39d) y en un 3% en el período global de cebo con niveles mayores de NDFS. El consumo medio diario durante la fase post-destete y durante todo el período de cebo, tendió a aumen tar (P≤0.079) con niveles mayores de FNDS, sin embargo no se apreció efecto sobre la ganancia media diaria (P≥0.15). En conclusión, el incremento del nivel de fibra soluble en el pienso parece resultar beneficioso para la salud del animal ya que mejora la integridad de la mucosa, y reduce la frecuencia de detección de potenciales patógenos como C. perfringens y Campylobacter spp. Conforme a estos resultados, debería tenerse en cuenta el contenido en fibra soluble en la formulación de piensos de conejos en la fase post-destete. El objetivo del segundo experimento fue determinar el efecto de la fuente de almidón sobre la digestión, la microbiota intestinal y los rendimientos productivos en conejos destetados con 25 días de edad. Se formularon tres piensos isonutritivos en los que se modificaron las principales fuentes de almidón: trigo crudo, trigo cocido y una combinación de trigo y arroz cocido. Dos grupos de 99 y 193 animales se destetaron con 25 días de edad. El primero de ellos se utilizó para la determinación de los parámetros productivos conforme al mismo protocolo seguido en el experimento anterior. El segundo de los grupos se utilizó para la determinación de la digestibilidad fecal de 32 a 35 d, la digestibilidad ileal aparente (DIA) a 35 d, la morfología de la mucosa intestinal, los parámetros de fermentación cecal; así como, la caracterización de la microbiota intestinal. Se utilizaron además dos grupos adicionales de animales 384 (medicados) y 177 (no medicados) para estudiar el efecto de la suplementación con antibióticos en el agua de bebida sobre la mortalidad. El procesado térmico del trigo mejoró ligeramente la digestibilidad ileal del almidón (P=0.020) pero no modificó el flujo final de almidón que alcanzó el ciego, observándose una mayor frecuencia de detección de Campylobacter spp. y Ruminococcus spp. en ciego (P≤0.023), pero sin cambios a nivel ileal. El procesado térmico del trigo no afectó tampoco a los parámetros productivos, la mortalidad, la digestibilidad ileal y fecal o la morfología de la mucosa. La sustitución parcial del trigo cocido por arroz cocido, penalizó la digestibilidad ileal del almidón (P=0.020) e incrementó el flujo ileal de este nutriente al ciego (P=0.007). Sin embargo no afectó a la mortalidad, pese a que se detectaron cambios en la microbiota tanto a nivel ileal como cecal, disminuyendo la frecuencia de detección de Campylobacter spp. (en íleon y ciego), Helicobacter spp. (en íleon) y Ruminococcus spp (en ciego) e incrementando Bacteroides spp. (en ciego) (P≤0.046). El empleo de arroz cocido en las piensos post-destete no tuvieron efectos sobre los parámetros productivos, la mortalidad, la digestibilidad ileal y fecal a excepción del almidón, o la morfología de la mucosa. La suplementación con antibiótico redujo la fre cuencia de detección de la mayoría de las bacterias estudiadas (P≤0.048), sobre todo para Campylobacter spp., Clostridium perfringens y Propionibacterium spp. (P≤0.048), observándose un efecto mayor a nivel ileal que cecal, lo que se asoció a la bajada significativa (P<0.001) de la mortalidad. En conclusión, los resultados de este experimento indican que la fuente de almidón afecta a la microbiota intestinal pero no influiye sobre la salud del animal. En relación al procesado, el uso de trigo cocido junto con arroz cocido no mejora los resultados obtenidos con trigo duro, si bienserían necesarios más experimentos que confirmaran este punto. El último de los experimentos se centró en un aspecto metodológico. Dado que, los conejos destetados presentan un patrón digestivo diferente al de un animal adulto resultado de su inmadurez digestiva, el objetivo buscado era tratar de determinar el mejor procedimiento para la determinación de la digestibilidad fecal en los gazapos en la fase post-destete. Para tal fin se utilizaron 15 animales/tratamiento de tres camadas diferentes que se destetaron con 25 días, suministrándoles un pienso comercial de crecimiento-cebo. Se registró el consumo medio diario y la excreción diaria de heces desde el día 25 hasta el día 40 de edad para la determinación de la digestibilidad de la MS. La camada afectó al consumo medio diario y la excreción de heces (P=0.013 y 0.014, respectivamente), observándose una tendencia (P=0.061) en la digestibilidad. La edad afectó (P<0.001) a todos estos factores, incrementándose de manera más evidente la excreción que la ingestión de materia seca en la primera semana de vida, para aumentar de forma paralela a partir de la segunda. La correlación entre el consumo medio diario fue mayor con la excreción de heces del mismo día que con la del día siguiente, por lo que se utilizó el primero para la determinación de la digestibilidad de la MS (MSd). La MSd disminuyó de manera lineal hasta los 32 días de edad (2.17±0.25 unidades porcentuales por día), mientras que permaneció constante desde los 32 a los 40 días (69.4±0.47%). Por otro lado, la desviación estándar de la MSd se redujo cuando se incrementó el período de recogida de 2 a 6 días en un 54%. Conforme a los resultados obtenidos, se puede concluir que no es aconsejable comenzar las pruebas de digestibilidad antes de los 32 días de edad y que el número de animales necesario para detectar diferencias significativas entre tratamientos dependerá del período de recogida de heces. ABSTRACT The global aim of this thesis has been to study the effect of dietary carbohydrates on growth, performance, digestion and intestinal barrier in 25-d weaned rabbits. In addition there has also been studied which is the best period to determine the fecal digestibility after weaning. The first experiment focused on the effect of Neutral Detergent Soluble Fibre (NDSF) on gut barrier function, digestion, intestinal microbiota and growth performance n rabbits in the post-weaning period. Three isonutritive diets which only varied in the levels of soluble fiber were formulated such as it described as follows: a control diet (AH) containing 103 g of neutral detergent soluble fiber, including alfalfa as main source of fiber, was replaced by a mixture of beet and apple pulp (75-25) in the B-AP diet and, by a mix of oat hulls and soybean protein concentrate (88:12) in the OH diet, resulting 131 and 79 g of NDFS/kg of dry matter, respectively. Rabbits, weaned at 25 days of age, were fed the experimental diets up to 35 days of age, moment in which they were slaughtered for apparent ileal digestibility (AID) of dry matter (DM), crude protein (CP) and starch, mucosa morphology, sucrose activity, characterization of lamina propria lymphocytes and intestinal microbiota. To assess mucosal morphology, 19 suckling 35-d-old rabbits were also used. For mortality study, besides these animals, 118 additional rabbits per treatment were fed the experimental diets for two weeks period and thereafter received a commercial diet until 60 days of age. Rabbits were water medicated during the whole experimental period (100 ppm de apramicine sulphate and 120 ppm of tylosine tartrate). Level of soluble fiber improved all the parameters used for the characterization of the intestinal barrier condition. Villous height of the jejunal mucosa increased with dietary soluble fiber (P=0.001). Villous height of jejunal mucosa increased with dietary soluble fiber (P = 0.001). Rabbits fed the highest level of soluble fiber (BA-P diet) showed the highest villous height/crypth depth ratio (8.14; P = 0.001), sucrase specific activity (8671 μmol glucose/ g protein; P = 0.019), and the greatest ileal starch digestibility (96.8%; P = 0.002). The opposite effects were observed in rabbits fed decreased levels of soluble fiber (AH and OH diets; 4.70, 5,848 μmol of glucose/g of protein, as average, respectively). The lowest ileal starch digestibility was detected for animal fed OH diet (93.2%). Suckling rabbits of the same age showed a lower villous height/crypt depth ratio (6.70) compared with the B-AP diet group, but this ration was higher that the AH or OH diet groups. Lower levels of soluble fiber tended (P = 0.074) to increase the cellular immune response (CD8+ lymphocytes). Diet affected IL-2 production (CD25+, P = 0.029; CD5+CD25+, P = 0.057), with no clear relationship between soluble fiber and IL-2. The intestinal microbiota biodiversity was not affected by diets (P ≥ 0.38). Animals fed B-AP and AH diets had a reduced cecal frequency of detection compatible with Campylobacter spp. (20.3 vs. 37.8, P = 0.074), and Clostridium perfringens (4.3 vs. 17.6%, P = 0.047), compared with the OH diet group. Moreover, the mortality rates decreased from 14.4 (OH diet) to 5.1% (B-AP diet) with the increased presence of soluble fiber in the diet. Between 32 and 35 days of age, faecal apparent digestibility of dry matter (DM), gross energy (GE), crude protein (CP), neutral detergent fiber (NDF), acid detergent fiber (ADF) and starch was determined (14/diet). This group, plus another nine rabbits/diet were used to determine weight of stomach and caecum and their contents, cecal fermentation traits and similarity rate (SR) of intestinal microbiota. Besides, growth performance parameters (35 rabbits/diet) were studied during the whole fattening period, in animals consuming the experimental feed after the weaning up to 35 days of age and later on a commercial diet up animals reached 60 days of age. Increasing levels of Neutral Detergent Soluble Fiber improved faecal dry matter and energy digestibility (P<0.001). NDSF inclusion improved linearly weight of the caecal content (P=0.001) and the total gastrointestinal tract (P=0.008), and in the previous days to slaughter a linear decrease of daily feed intake in diet with highest level of soluble fiber was also observed. Stomach pH decreased linearly with increasing levels of NDFS (P≤0.041). No relation between NDSF level on pH, concentration and molar proportion of VFA was found. Treatments appeared to influence the similarity rate of microbiota, even higher to mother effect. These effects were higher in ileum than in caecum. A linear positive effect of feed efficiency was observed, which increased around 12% in the two weeks post-weaning (25-39d) and 3% in the whole fattening period between extreme diets with highest levels of soluble fiber. Average daily feed intake during the two weeks after weaning and in the whole fattening period, tended (P≤0.079) to increase with highest levels of NDSF; although there were no effect on daily weight gain (≥0.15). In conclusion, an increase of soluble fiber in the feed seems to be beneficial for animal health, due to improve mucose integrity and reduce detection frequency of those poten tial pathogens like C. perfringens and Campylobacter spp. According to these results, level of soluble fiber should be taking care in feed rabbit formulation in the post-weaning period. The objective of the second experiment was to determine the effect of source of starch on digestion, intestinal microbiota and growth performance in twenty-five-day old weaned rabbits. To accomplish with this aim three iso-nutritive diets were formulated with different source of starch: raw wheat, boiled wheat and a combination of boiled wheat and boiled rice. Two groups of 99 and 193 rabbits were weaned at 25 days of age. The first group was used for growth performance determination following the same protocol than in previous experiment. The second group was used to determine faecal digestibility from 32 to 35 d, apparent ileal digestibility (AID) at 35 d, jejunal mucosa morphology, caecal fermentation traits and characterization of intestinal microbiota. For mortality, two additional groups of 384 (medicated) and 177 (not medicated) were used in order to study the effect of antibiotic water supply supplementation. Heat processing of starch slightly improved ileal digestibility of starch (P=0.020) but did not modify the flow of starch to the caecum. An increase in frequency of detection of Campylobacter spp. y Ruminococcus spp. was observed in the caecum (P≤0.023), with no changes at ileal level. Heat processing of wheat did not modify growth performance, mortality, ileal or faecal digestibility and mucosa morphology. Partial substitution of boiled wheat for boiled rice in the diet impaired ileal starch digestibility (P=0.020) and increased the ileal flow of this nutrient to the caecum (P=0.007). However, it did not affect mortality rate, although changes in the ileal and caecal intestinal microbiota were detected, decreasing the frequency of detection of Campylobacter spp. (both ileum and caecum), Helicobacter spp. (at ileum) and Ruminococcus spp (at caecum) and increasing the Bacteroides spp. (at caecum) (P≤0.046). The effect of boiled rice supplementation did not alter growth performance, mortality, ileal or faecal digestibility of other nutrients than starch, and mucosa morphology. Medication of rabbits reduced the ileal frequency of detection of most bacteria studied (P≤0.048), especially for Campylobacter spp., Clostridium perfringens y Propionibacterium spp. (P≤0.048), resulting the effect higher at ileal than caecal level and relating it with a strong reduction of mortality rate (P<0.001). In conclusion, the results of this experiment make think that the source of starch affects the intestinal microbiota but they do not seem to influence animal health. In relation to the effect of heat processed the use of cooked wheat or cooked rice it does not seem to im prove the results obtained with hard wheat, but there would be necessary more experiments that were confirming this point. The last experiment focused on a methodological aspect. Considering that, weaned rabbits have a different digestive pattern than older animals due to their digestive immaturity; the fixed objective was to determine the best procedure for faecal digestibility determination in young rabbits in the post-weaning period. Fifteen rabbits from 5 different litters were weaned at 25 days of age and fed with a commercial feed. Feed intake and faeces excretion were recorded daily from 25 to 40 days of age for dry matter digestibility (DMd) determination. Litter affected daily DM intake and excretion (P=0.013 y 0.014, respectively) and tended to affect DMd (P=0.061). Age affected all these factors (P<0.001), but ingestion increased slowly than dry matter excretion during the first week buth they evolved similarly in the second week. The correlation between daily feed intakes was higher with the faeces excretion of the day than with faeces excretion of the next day, and the first values were used to determine daily DMd. The DMd decreased linearly from weaning to 32 d of age (2.17±0.25 percentage units per day), whereas from 32 to 40 d remained constant (69.4±0.47%). On the other hand, average standard deviation of DMd decreased by 54% when the length of collection period increased from 2 to 6d. Consequently to the obtained results, it could be concluded that it would not be advisable to start digestibility trials before the 32 days of age and that the number of animals required to detect a significant difference among means would depend on the collection period.

Structural and morphological studies on wet-etched InAlGaN barrier HEMT structures

The quaternary nitride-based high electron mobility transistor (HEMT) has been recently a focus of interest because of the possibility to grow lattice-matched barrier to GaN and tune the barrier bandgap at the same time.

Investigation of AlInN barrier ISFET structures with GaN capping for pH detection

In the last decade the interest in nitride-based sensors (gas, ions...) and bio-sensors is increased. In the case of ion sensitive FET (ISFET), gate voltages induced by ions adsorbed onto the gate region modulate the source-drain currents.

Propagation losses by tunnelling in barrier optical waveguides

An analytical model is proposed in order to estimate the optical propagation losses due to tunnelling in barrier waveguides. The results are validated by means of a beam propagation method (BPM) simulations for different waveguides conditions.

Choroid plexus epithelial expression of MDR1 P glycoprotein and multidrug resistance-associated protein contribute to the blood–cerebrospinal-fluid drug-permeability barrier

The blood–brain barrier and a blood–cerebrospinal-fluid (CSF) barrier function together to isolate the brain from circulating drugs, toxins, and xenobiotics. The blood–CSF drug-permeability barrier is localized to the epithelium of the choroid plexus (CP). However, the molecular mechanisms regulating drug permeability across the CP epithelium are defined poorly. Herein, we describe a drug-permeability barrier in human and rodent CP mediated by epithelial-specific expression of the MDR1 (multidrug resistance) P glycoprotein (Pgp) and the multidrug resistance-associated protein (MRP). Noninvasive single-photon-emission computed tomography with 99mTc-sestamibi, a membrane-permeant radiopharmaceutical whose transport is mediated by both Pgp and MRP, shows a large blood-to-CSF concentration gradient across intact CP epithelium in humans in vivo. In rats, pharmacokinetic analysis with 99mTc-sestamibi determined the concentration gradient to be greater than 100-fold. In membrane fractions of isolated native CP from rat, mouse, and human, the 170-kDa Pgp and 190-kDa MRP are identified readily. Furthermore, the murine proteins are absent in CP isolated from their respective mdr1a/1b(−/−) and mrp(−/−) gene knockout littermates. As determined by immunohistochemical and drug-transport analysis of native CP and polarized epithelial cell cultures derived from neonatal rat CP, Pgp localizes subapically, conferring an apical-to-basal transepithelial permeation barrier to radiolabeled drugs. Conversely, MRP localizes basolaterally, conferring an opposing basal-to-apical drug-permeation barrier. Together, these transporters may coordinate secretion and reabsorption of natural product substrates and therapeutic drugs, including chemotherapeutic agents, antipsychotics, and HIV protease inhibitors, into and out of the central nervous system.

The neuroprotective agent SR 57746A abrogates experimental autoimmune encephalomyelitis and impairs associated blood–brain barrier disruption: Implications for multiple sclerosis treatment

Experimental autoimmune encephalomyelitis (EAE) is a T cell autoimmune disorder that is a widely used animal model for multiple sclerosis (MS) and, as in MS, clinical signs of EAE are associated with blood–brain barrier (BBB) disruption. SR 57746A, a nonpeptide drug without classical immunosuppressive properties, efficiently protected the BBB and impaired intrathecal IgG synthesis (two conventional markers of MS exacerbation) and consequently suppressed EAE clinical signs. This compound inhibited EAE-induced spinal cord mononuclear cell invasion and normalized tumor necrosis factor α and IFN-γ mRNA expression within the spinal cord. These data suggested that pharmacological intervention aimed at inhibiting proinflammatory cytokine expression within the central nervous system provided protection against BBB disruption, the first clinical sign of EAE and probably the key point of acute MS attacks. This finding could lead to the development of a new class of compounds for oral therapy of MS, as a supplement to immunosuppressive agents.

Deltorphin transport across the blood–brain barrier

In vivo antinociception studies demonstrate that deltorphins are opioid peptides with an unusually high blood–brain barrier penetration rate. In vitro, isolated bovine brain microvessels can take up deltorphins through a saturable nonconcentrative permeation system, which is apparently distinct from previously described systems involved in the transport of neutral amino acids or of enkephalins. Removing Na+ ions from the incubation medium decreases the carrier affinity for deltorphins (−25%), but does not affect the Vmax value of the transport. The nonselective opiate antagonist naloxone inhibits deltorphin uptake by brain microvessels, but neither the selective δ-opioid antagonist naltrindole nor a number of opioid peptides with different affinities for δ- or μ-opioid receptors compete with deltorphins for the transport. Binding studies demonstrate that μ-, δ-, and κ-opioid receptors are undetectable in the microvessel preparation. Preloading of the microvessels with l-glutamine results in a transient stimulation of deltorphin uptake. Glutamine-accelerated deltorphin uptake correlates to the rate of glutamine efflux from the microvessels and is abolished by naloxone.

On the electronic nature of low-barrier hydrogen bonds in enzymatic reactions

The electronic nature of low-barrier hydrogen bonds (LBHBs) in enzymatic reactions is discussed based on combined low temperature neutron and x-ray diffraction experiments and on high level ab initio calculations by using the model substrate benzoylacetone. This molecule has a LBHB, as the intramolecular hydrogen bond is described by a double-well potential with a small barrier for hydrogen transfer. From an “atoms in molecules” analysis of the electron density, it is found that the hydrogen atom is stabilized by covalent bonds to both oxygens. Large atomic partial charges on the hydrogen-bonded atoms are found experimentally and theoretically. Therefore, the hydrogen bond gains stabilization from both covalency and from the normal electrostatic interactions found for long, weak hydrogen bonds. Based on comparisons with other systems having short-strong hydrogen bonds or LBHBs, it is proposed that all short-strong and LBHB systems possess similar electronic features of the hydrogen-bonded region, namely polar covalent bonds between the hydrogen atom and both heteroatoms in question.

Prion protein NMR structure and species barrier for prion diseases

The structural basis of species specificity of transmissible spongiform encephalopathies, such as bovine spongiform encephalopathy or “mad cow disease” and Creutzfeldt–Jakob disease in humans, has been investigated using the refined NMR structure of the C-terminal domain of the mouse prion protein with residues 121–231. A database search for mammalian prion proteins yielded 23 different sequences for the fragment 124–226, which display a high degree of sequence identity and show relevant amino acid substitutions in only 18 of the 103 positions. Except for a unique isolated negative surface charge in the bovine protein, the amino acid differences are clustered in three distinct regions of the three-dimensional structure of the cellular form of the prion protein. Two of these regions represent potential species-dependent surface recognition sites for protein–protein interactions, which have independently been implicated from in vitro and in vivo studies of prion protein transformation. The third region consists of a cluster of interior hydrophobic side chains that may affect prion protein transformation at later stages, after initial conformational changes in the cellular protein.

Functional breakdown of the lipid bilayer of the myelin membrane in central and peripheral nervous system by disrupted galactocerebroside synthesis

The lipid bilayer of the myelin membrane of the central nervous system (CNS) and the peripheral nervous system (PNS) contains the oligodendrocyte- and Schwann cell-specific glycosphingolipids galactocerebrosides (GalC) and GalC-derived sulfatides (sGalC). We have generated a UDP-galactose ceramide galactosyltransferase (CGT) null mutant mouse (cgt−/−) with CNS and PNS myelin completely depleted of GalC and derived sGalC. Oligodendrocytes and Schwann cells are unable to restore the structure and function of these galactosphingolipids to maintain the insulator function of the membrane bilayer. The velocity of nerve conduction of homozygous cgt−/− mice is reduced to that of unmyelinated axons. This indicates a severely altered ion permeability of the lipid bilayer. GalC and sGalC are essential for the unperturbed lipid bilayer of the myelin membrane of CNS and PNS. The severe dysmyelinosis leads to death of the cgt−/− mouse at the end of the myelination period.

Fractionation factors and activation energies for exchange of the low barrier hydrogen bonding proton in peptidyl trifluoromethyl ketone complexes of chymotrypsin

NMR investigations have been carried out of complexes between bovine chymotrypsin Aα and a series of four peptidyl trifluoromethyl ketones, listed here in order of increasing affinity for chymotrypsin: N-Acetyl-l-Phe-CF3, N-Acetyl-Gly-l-Phe-CF3, N-Acetyl-l-Val-l-Phe-CF3, and N-Acetyl-l-Leu-l-Phe-CF3. The D/H fractionation factors (φ) for the hydrogen in the H-bond between His 57 and Asp 102 (His 57-Hδ1) in these four complexes at 5°C were in the range φ = 0.32–0.43, expected for a low-barrier hydrogen bond. For this series of complexes, measurements also were made of the chemical shifts of His 57-Hɛ1 (δ2,2-dimethylsilapentane-5-sulfonic acid 8.97–9.18), the exchange rate of the His 57-Hδ1 proton with bulk water protons (284–12.4 s−1), and the activation enthalpies for this hydrogen exchange (14.7–19.4 kcal⋅mol−1). It was found that the previously noted correlations between the inhibition constants (Ki 170–1.2 μM) and the chemical shifts of His 57-Hδ1 (δ2,2-dimethylsilapentane-5-sulfonic acid 18.61–18.95) for this series of peptidyl trifluoromethyl ketones with chymotrypsin [Lin, J., Cassidy, C. S. & Frey, P. A. (1998) Biochemistry 37, 11940–11948] could be extended to include the fractionation factors, hydrogen exchange rates, and hydrogen exchange activation enthalpies. The results support the proposal of low barrier hydrogen bond-facilitated general base catalysis in the addition of Ser 195 to the peptidyl carbonyl group of substrates in the mechanism of chymotrypsin-catalyzed peptide hydrolysis. Trends in the enthalpies for hydrogen exchange and the fractionation factors are consistent with a strong, double-minimum or single-well potential hydrogen bond in the strongest complexes. The lifetimes of His 57-Hδ1, which is solvent shielded in these complexes, track the strength of the hydrogen bond. Because these lifetimes are orders of magnitude shorter than those of the complexes themselves, the enzyme must have a pathway for hydrogen exchange at this site that is independent of dissociation of the complexes.

Absence of a barrier to backwards rotation of the bacterial flagellar motor demonstrated with optical tweezers

A cell of the bacterium Escherichia coli was tethered covalently to a glass coverslip by a single flagellum, and its rotation was stopped by using optical tweezers. The tweezers acted directly on the cell body or indirectly, via a trapped polystyrene bead. The torque generated by the flagellar motor was determined by measuring the displacement of the laser beam on a quadrant photodiode. The coverslip was mounted on a computer-controlled piezo-electric stage that moved the tether point in a circle around the center of the trap so that the speed of rotation of the motor could be varied. The motor generated ≈4500 pN nm of torque at all angles, regardless of whether it was stalled, allowed to rotate very slowly forwards, or driven very slowly backwards. This argues against models of motor function in which rotation is tightly coupled to proton transit and back-transport of protons is severely limited.

Purified hematopoietic stem cells without facilitating cells can repopulate fully allogeneic recipients across entire major histocompatibility complex transplantation barrier in mice

We report herein the successful long term engraftment of highly purified hematopoietic stem cells (HSCs) without any facilitating cells in fully allogeneic recipient mice across the entire major histocompatibility complex (MHC) transplantation barrier. This finding challenges the assumption that highly purified marrow HSCs alone cannot produce long-lived allogeneic bone marrow chimeras across the MHC barrier. In the present experiments, 1 × 105 HSCs from 5-fluorouracil (5-FU)-treated donors, without any facilitating cells, have been found to repopulate lethally irradiated fully allogeneic recipients. Low density, lineage-negative (CD4−, CD8−, B220−, Mac-1−, Gr-1−), CD71-negative, class I highly positive, FACS-sorted cells from 5-FU-treated C57BL/6 (B6) donor mice were transplanted into lethally irradiated BALB/c recipients. (BALB/c → BALB/c) → BALB/c T cell-depleted marrow cells used as compromised cells were also transplanted into the recipients to permit experiments to be pursued over a long period of time. Cells of donor origin in all recognized lineages of hematopoietic cells developed in these allogeneic chimeras. One thousand HSCs were sufficient to repopulate hemiallogeneic recipients, but 1 × 104 HSCs alone from 5-FU-treated donors failed to repopulate the fully allogeneic recipients. Transplantation of primary marrow stromal cells or bones of the donor strain into recipient, together with 1 × 104 HSCs, also failed to reconstitute fully allogeneic recipients. Suppression of resistance of recipients by thymectomy or injections of granulocyte colony-stimulating factor before stem cell transplantation enhanced the engraftment of allogeneic HSCs. Our experiments show that reconstitution of all lymphohematopoietic lineages across the entire MHC transplantation barriers may be achieved by transplanting allogeneic HSCs alone, without any facilitating cells, as long as a sufficient number of HSCs is transplanted.

Regulation of the Actin Cytoskeleton by Thrombin in Human Endothelial Cells: Role of Rho Proteins in Endothelial Barrier Function

Endothelial barrier function is regulated at the cellular level by cytoskeletal-dependent anchoring and retracting forces. In the present study we have examined the signal transduction pathways underlying agonist-stimulated reorganization of the actin cytoskeleton in human umbilical vein endothelial cells. Receptor activation by thrombin, or the thrombin receptor (proteinase-activated receptor 1) agonist peptide, leads to an early increase in stress fiber formation followed by cortical actin accumulation and cell rounding. Selective inhibition of thrombin-stimulated signaling systems, including Gi/o (pertussis toxin sensitive), p42/p44, and p38 MAP kinase cascades, Src family kinases, PI-3 kinase, or S6 kinase pathways had no effect on the thrombin response. In contrast, staurosporine and KT5926, an inhibitor of myosin light chain kinase, effectively blocked thrombin-induced cell rounding and retraction. The contribution of Rho to these effects was analyzed by using bacterial toxins that either activate or inhibit the GTPase. Escherichia coli cytotoxic necrotizing factor 1, an activator of Rho, induced the appearance of dense actin cables across cells without perturbing monolayer integrity. Accordingly, lysophosphatidic acid, an activator of Rho-dependent stress fiber formation in fibroblasts, led to reorganization of polymerized actin into stress fibers but failed to induce cell rounding. Inhibition of Rho with Clostridium botulinum exoenzyme C3 fused to the B fragment of diphtheria toxin caused loss of stress fibers with only partial attenuation of thrombin-induced cell rounding. The implication of Rac and Cdc42 was analyzed in transient transfection experiments using either constitutively active (V12) or dominant-interfering (N17) mutants. Expression of RacV12 mimicked the effect of thrombin on cell rounding, and RacN17 blocked the response to thrombin, whereas Cdc42 mutants were without effect. These observations suggest that Rho is involved in the maintenance of endothelial barrier function and Rac participates in cytoskeletal remodeling by thrombin in human umbilical vein endothelial cells.