Pharmaceutical nanocrystals confined in porous host systems--interfacial effects and amorphous interphases.

DSC and NMR results prove the existence of an amorphous acetaminophen nanolayer between acetaminophen nanocrystals (form I and form III) and pore walls in controlled porous glasses. This nanolayer controls the surface energy. This finding is highly relevant for approaches towards crystal engineering in nanopores and indicates similarities to the situation during the early stages of crystallization in polymorphic bulk samples.

Ultra-thin porous glass membranes--an innovative material for the immobilization of active species for optical chemosensors.

In addition to polymers, porous glasses can be used for the immobilization of indicators, chromoionophores or enzymes. Advantages of these materials include, among others, the photochemical and thermal stability. Porous glass membranes (CPG) based on phase-separated alkali borosilicate glasses with thicknesses of 250-300 µm and dimensions of approximately 9-13 mm² were used in this work. The average pore diameter was found to be between 12 and 112 nm. Initially, the membrane permeability for water was determined. Furthermore, the absorption spectra for the water-soaked membranes were recorded optically. CPG membranes which are pH-sensitive were prepared based on the covalent immobilization of thymol blue and a derivative of styryl acridine. In each case, the absorption spectra of the immobilized indicators are shown. The t90-times vary between 4 and 20 min and were determined for the thermodynamic equilibrium. The influence of the ionic strength on the characteristic curve is discussed and detailed results are given. After the storage time of about 900 days a pH-sensitivity for a CPG membrane styryl acridine derivative sample was still detectable.

Size-dependent growth of polymorphs in nanopores and Ostwald's step rule of stages.

More than 100 years after Ostwald postulated his step rule of stages, predictive understanding as to early crystallization stages of polymorphic materials is still premature. We studied crystallization of the polymorphic pharmaceutical acetaminophen in nanoporous glasses as a model for early stages of bulk crystallization since the surface energy significantly contributes to the total Gibbs free energy of nanosized crystals in both cases. Systematic studies of transitions between different polymorphs inside nanoporous glasses show that the thermodynamic stability of the polymorphs depends on the crystal size. Accordingly, the transient occurrence of different polymorphs during crystal growth in bulk systems can be related to surface energy contributions to the total Gibbs free energy of the developing crystals. In nanosized early-stage crystals with high surface-to-volume ratios other polymorphs may be stable than in large crystals with low surface-to-volume ratios. Improved control of the crystallization of polymorphic materials by imposing well-defined confinement is a promising strategy to tailor release of polymorphic drugs and to optimize optical, electronic, magnetic and ferroelectric properties of polymorphic materials.

Cavitation and pore blocking in nanoporous glasses.

In gas adsorption studies, porous glasses are frequently referred to as model materials for highly disordered mesopore systems. Numerous works suggest that an accurate interpretation of physisorption isotherms requires a complete understanding of network effects upon adsorption and desorption, respectively. The present article deals with nitrogen and argon adsorption at different temperatures (77 and 87 K) performed on a series of novel nanoporous glasses (NPG) with different mean pore widths. NPG samples contain smaller mesopores and significantly higher microporosity than porous Vycor glass or controlled pore glass. Since the mean pore width of NPG can be tuned sensitively, the evolution of adsorption characteristics with respect to a broadening pore network can be investigated starting from the narrowest nanopore width. With an increasing mean pore width, a H2-type hysteresis develops gradually which finally transforms into a H1-type. In this connection, a transition from a cavitation-induced desorption toward desorption controlled by pore blocking can be observed. Furthermore, we find concrete hints for a pore size dependence of the relative pressure of cavitation in highly disordered pore systems. By comparing nitrogen and argon adsorption, a comprehensive insight into adsorption mechanisms in novel disordered materials is provided.

Fitase na dieta de alevinos de carpa húngara: desempenho e características de carcaça / Phytase in diet of common carp fingerlings: produtive performance and carcass traits

Foram utilizados 240 alevinos de carpa húngara, com peso médio de 3,42±0,32g, distribuídos em delineamento inteiramente ao acaso, com quatro tratamentos e quatro repetições. A dieta basal foi formulada com ingredientes de origem vegetal (36 por cento de proteína bruta e 3.200kcal de energia digestível). Os tratamentos corresponderam a quatro níveis de fitase ativa (UF) por kg na dieta: zero (controle), 500, 1000 e 1500UF/kg. A cada 15 dias, foi realizada uma biometria e, ao término do período experimental (45 dias), 5 por cento dos animais foram eutanasiados para determinação do rendimento de carcaça e composição químico-bromatológica. Os parâmetros de desempenho avaliados foram: peso final, ganho de peso, comprimento total, taxa de crescimento específico e sobrevivência. A suplementação de fitase não influiu no desempenho produtivo, entretanto melhorou significativamente as características da carcaça.

A total of 240 fingerlings, with initial average weight 3.42±0.32g, were distributed in a completely randomized experimental design, with four treatments and four replicates. Basal diet consisted of vegetal ingredients (36 percent crude protein and 3,220kcal of digestible energy). Treatments corresponded to four levels of active phytase (UF) per kg of diet: zero (control), 500, 1,000, and 1,500. Every 15 days, a biometry was made and at the end of the experimental period (45 days) 5 percent of animals were euthanized to determine carcass yield and chemical-bromatologic composition. The following variables were evaluated: final weight, weight gain, total length, specific growth rate, and survial. There was no effect of phytase on productive performance; however, it significantly improved carcass characteristics.

Further advancements in predicting adsorption equilibria using excess formalism: calculation of adsorption excesses at the liquid/solid interface.

Prediction of adsorption equilibria for ternary liquid mixtures on solid surfaces by means of adsorption data for the corresponding three binary liquid mixtures can be improved by combining the thermodynamic excess formalism with geometrical models. This new strategy for the prediction of excess adsorption isotherms is examined for four ternary adsorption systems ranging from ideal to highly non-ideal ternary mixtures. The predicted isotherms are discussed and compared with experimental ones as well as with those obtained for a model based on the absolute quantities. The results confirm: (i) superiority of predicting adsorption in terms of excess quantities, and (ii) utility of geometrical models for constructing ternary molar compositions on the basis of binary ones to predict equilibria not only for liquid mixtures alone but also for adsorption of liquid mixtures on solid surfaces.

Manipulating the crystalline state of pharmaceuticals by nanoconfinement.

We show that nanoconfinement is a handle to rationally produce and stabilize otherwise metastable or transient polymorphs of pharmaceuticals, as required for controllable and efficient drug delivery. The systematic investigation of crystallization under confinement unveils thermodynamic properties of metastable polymorphs not accessible otherwise and may enhance the understanding of the crystallization behavior of pharmaceuticals in general. As an example in this case, we studied acetaminophen confined to inexpensive and biocompatible nanoporous host systems. Calorimetric and X-ray scattering data clearly evidence that either the stable polymorph form I or the metastable polymorph form III can be stabilized in high yields. Thermodynamic parameters for form III of acetaminophen are reported, and strategies to manipulate the crystalline state in pores by thermal treatments are presented.

Global gene expression analysis reveals a role for the alpha 1 integrin in renal pathogenesis.

Kidney fibrosis is the hallmark of most types of progressive kidney disease, including the genetic disorder Alport's syndrome. We undertook gene expression analysis in Alport's syndrome mouse kidneys using microchip arrays to characterize the development of fibrosis. In addition to matrix and matrix-remodeling genes, consistent with interstitial fibrosis, macrophage-related genes show elevated expression levels in Alport's syndrome kidneys. Immunohistochemical analysis of kidney sections illustrated that macrophages as well as myofibroblasts accumulate in the tubular interstitium. Deletion of alpha(1) integrin results in decreased accumulation of both myofibroblasts and macrophages in the tubular interstitium in Alport's syndrome mice and delays disease progression. Transforming growth factor beta antagonism, although reducing interstitial fibrosis, does not limit macrophage accumulation in the tubular interstitium and disease progression. In this study, we identified previously overlooked inflammatory events that occur in the tubulointerstitial region. We propose that in addition to the previously suggested role for the alpha(1)beta(1) integrin in mesangial expansion and abnormal laminin deposition, this integrin may be critical for monocyte accumulation that, in turn, may lead directly to renal failure. Our gene expression and immunohistochemical data indicate that macrophage accumulation is dependent on alpha(1) integrin expression on the macrophage cell surface and that anti-alpha(1) integrin strategies may be employed as therapeutics in the treatment of chronic inflammatory and fibrotic diseases.

Kinetic analysis of the cyclin-dependent kinase-activating kinase (Cak1p) from budding yeast.

Cak1p, the Cyclin-dependent kinase-activating kinase from budding yeast, is an unusual protein kinase that lacks many of the highly conserved motifs observed among members of the protein kinase superfamily. Cak1p phosphorylates and activates Cdc28p, the major cyclin-dependent kinase (CDK) in yeast, and is thereby required for passage through the yeast cell cycle. In this paper, we explore the kinetics of CDK phosphorylation by Cak1p, and we examine the role of the catalytic step in the reaction mechanism. Cak1p proceeds by a sequential reaction mechanism, binding to both ATP and CDK2 with reasonable affinities, exhibiting K(d) values of 7.2 and 0.6 microm, respectively. Interestingly, these values are approximately the same as the K(M) values, indicating that the binding of substrates is fast with respect to catalysis and that the most likely reaction mechanism is rapid equilibrium random. Cak1p is a slow enzyme, with a catalytic rate of only 4.3 min(-)(1). The absence of a burst phase indicates that product release is not rate-limiting. This result, and a solvent isotope effect, suggests that a catalytic step is rate-limiting.

The CDK-activating kinase (Cak1p) from budding yeast has an unusual ATP-binding pocket.

Cak1p is an essential protein kinase that phosphorylates and thereby activates the major cyclin-dependent kinase in budding yeast, Cdc28p. The sequence of Cak1p differs from other members of the protein kinase superfamily in several conserved regions. Cak1p lacks the highly conserved glycine loop motif (GXGXXG) that is found in the nucleotide binding fold of virtually all protein kinases and also lacks a number of conserved amino acids found at sites throughout the protein kinase core sequence. We have used kinetic and mutagenic analyses to investigate whether these sequence differences affect the nucleotide-binding properties of Cak1p. Although Cak1p differs dramatically from other protein kinases, it binds ATP with a reasonable affinity, with a KM of 4.8 microM. Mutations of the putative invariant lysine in Cak1p (Lys-31), homologous to a residue required for activity in virtually all protein kinases and that interacts with the ATP phosphates, moderately reduced the ability of Cak1p to bind ATP but did not dramatically affect the catalytic rate of the kinase. Similarly, Cak1p is insensitive to the ATP analog 5'-fluorosulfonylbenzoyladenosine, which inhibits most protein kinases through covalent modification of the invariant lysine. We found that Cak1p is tolerant of mutations within its glycine loop region. Remarkably, Cak1p remains functional even following truncation of its first 31 amino acids, including the glycine loop region and the invariant lysine. We conclude that the Cak1p nucleotide-binding pocket differs significantly from those of most other protein kinases and therefore might provide a specific target for an inhibitory drug.

Localization and regulation of the cdk-activating kinase (Cak1p) from budding yeast.

Eukaryotic cell cycles are controlled by the activities of cyclin-dependent kinases (cdks). The major cdk in budding yeast, Saccharomyces cerevisiae, is Cdc28p. Activation of Cdc28p requires phosphorylation on threonine 169 and binding to a cyclin. Thr-169 is phosphorylated by the cdk-activating kinase (CAK), Cak1p, which was recently identified as the physiological CAK in budding yeast. Here we present our further characterization of yeast Cak1p. We have found that Cak1p is dispersed throughout the cell as shown by immunofluorescence; biochemical subcellular fractionation confirmed that most of the Cak1p is found in the cytoplasm. Cak1p is a monomeric enzyme in crude yeast lysates. Mutagenesis of potential sites of activating phosphorylation had little effect on the activity of Cak1p in vitro or in vivo. Furthermore, Cak1p contains no posttranslational modifications detectable by two-dimensional isoelectric focusing. We found that Cak1p is a stable protein during exponential growth but that its expression decreases considerably when cells enter stationary phase. In contrast, Cak1p levels oscillate dramatically during meiosis, reflecting regulation at both the transcriptional and post-translational level. The localization and regulation of Cak1p are in contrast to those of the known vertebrate CAK, p40(MO15).