Structural and theoretical-experimental physicochemical study of trimethoprim/randomly methylated-β-cyclodextrin binary system

Here we report the structural characterization, physicochemical study and molecular modeling of the inclusion complex of trimethoprim in randomly methylated beta-cyclodextrin. The phase-solubility diagram obtained at pH 7.0 exhibited a linear behavior for the RAMEB concentrations studied suggesting a 1:1 stoichiometry and absence of aggregation in solution. From stoichiometric determination by the continuous variation method we confirmed a 1:1 stoichiometry. To make a detailed characterization of the inclusion mode, spectroscopic measurements by infrared and 1D and 2D ¹H NMR spectroscopy provided evidence that the inclusion mode is characterized by inclusion of the trimethoxyphenyl ring in the cavity; interactions with methyl groups located in the border of the cavity were also detected. The structure proposed was also confirmed by semiempirical molecular modeling.     

The enhancement of the oral bioavailability of γ-tocotrienol in mice by γ-cyclodextrin inclusion

Cyclodextrin (CD) is widely used in the pharmaceutical and nutritional fields to form an inclusion complex with lipophilic compounds for the improvement of their aqueous solubility, stability and diffusibility under physiological conditions. In this study, we investigated the effect of the γ-tocotrienol (γT3) inclusion complex with CD on its oral bioavailability. Five-week-old C57BL6 mice were fed a vitamin E-free diet for 28 days, followed by the oral administration of 2.79 mg of γT3-rich fraction (TRF) extracted from rice bran or the equivalent dose (14.5 mg) of a CD inclusion complex with TRF (TRF/CD). The levels of γT3 in sequentially collected plasma were determined by LC-MS/MS. The pharmacokinetic study revealed that the plasma concentrations of γT3 were increased and peaked at 6 or 3 h after the oral administration of TRF or TRF/CD, respectively (C_max values of 7.9±3.3 or 11.4±4.5 μM, respectively). The area under the curve of plasma γT3 concentration also showed a 1.4-fold increase in the group administered with TRF/CD compared with the TRF-only group. Furthermore, the mice that had received the TRF/CD tended to reduce the endotoxin shock induced by injection with lethal amounts of Escherichia coli lipopolysaccharide, compared with the mice that had received TRF alone. Taken together, our results suggest that the CD inclusion improved γT3 bioavailability, resulting in the enhancement of γT3 physiological activity, which would be a useful approach for the nutrition delivery system.     

Diclofenac-β-cyclodextrin binary systems: Physicochemical characterization and in vitro dissolution and diffusion studies

The aim of this work was to study the influence of β-cyclodextrin (β-CD) on the biopharmaceutic properties of diclofenac (DCF). To this purpose the physicochemical characterization of diclofenac-β-cyclodextrin binary systems was performed both in solution and solid state. Solid phase characterization was performed using differential scanning calorimetry (DSC), powder x-ray diffractometry (XRD), and Fourier transform infrared spectroscopy (FTIR). Phase solubility analyses, and in vitro permeation experiments through a synthetic membrane were performed in solution. Moreover, DCF/β-CD interactions were studied in DMSO by¹H nuclear magnetic resonance (NMR) spectroscopy. The effects of different preparation methods and drug-to-β-CD molar ratios were also evaluated. Phase solubility studies revealed 1∶1 M complexation of DCF when the freeze-drying method was used for the preparation of the binary system. The true inclusion for the freeze-dried binary system was confirmed by¹H NMR spectroscopy, DSC, powder XRD, and IR studies. The dissolution study revealed that the drug dissolution rate was improved by the presence of CDs and the highest and promptest release was obtained with the freeze-dried binary system. Diffusion experiments through a silicone membrane showed that DCF diffusion was higher from the saturated drug solution (control) than the freeze-dried inclusion complexes, prepared using different DCF-β-CD molar ratios. However, the presence of the inclusion complex was able to stabilize the system giving rise to a more regular diffusion profile. Published: October 22, 2005     

Investigation on a host-guest inclusion system by β-cyclodextrin derivative and its analytical application

The host-guest inclusion system of ethyl substituted β-cyclodextrin (DE-β-CD) with mangiferin (MA) was investigated by fluorescence spectra in solution. The results showed that the MA was encapsulated in the DE-β-CD’s cavity to form a 2:1 stoichiometry host-guest inclusion complex (DE-β-CD/MA) and the inclusion constant (K = 3.04 × 10⁶ L²/mol²) was confirmed by the typical double reciprocal plots. Furthermore, several experimental conditions were optimized in order to obtain the maximum fluorescence signal. In addition, the thermodynamic parameters, Gibbs free energy (ΔG°), enthalpy change (ΔH°) and entropy change (ΔS°) of DE-β-CD/MA were obtained by the Van’t Hoff equation. A spectrofluorimetric method for the determination of MA in solution in the presence of DE-β-CD was developed based on the remarkable enhancement of the fluorescence intensity of MA. The linear range was 2.00 × 10⁻⁸-7.00 × 10⁻⁶ mol/L and the detection limit was 4.05 × 10⁻⁹ mol/L. The proposed method was successfully applied to the analysis of MA in serum with the satisfactory result.     

The probability of conception after discontinuance of oral contraception: A note on “oral contraception,coital frequency,and the time required to conceive,” by Westoff,Bumpass, and Ryder

The apolipoprotein E (ApoE) gene, which has three common alleles (?2, ?3, and ?4), has been linked to a number of health outcomes and longevity. The ?2 allele has been reported to have neuroprotective effects, whereas the ?4 allele has been shown to be a risk factor for cardiovascular disease and Alzheimer's disease in various populations. The relationships between ApoE and mortality and ApoE and physical function, however, are not clear-cut. We used the Social Environment and Biomarkers of Aging Study (SEBAS) to examine the relationship between ApoE polymorphisms and physical and pulmonary function in approximately 1,000 Taiwanese adults aged 53 years and older in 2006. In the 2006 SEBAS wave, measures of physical function included self-reported difficulties with respect to activities of daily living (ADLs) and other physical function indicators, as well as performance-based measures of grip strength (kg), walking speed (m/s) over a distance of 3 m, and chair stand speed (stand/s). Peak expiratory flow (PEF; L/min) rate was also examined as an indicator of pulmonary function. We used logistic regression models to determine the association between ApoE and inability to complete each of the tests of physical and pulmonary function. These models revealed no significant association between ApoE carrier status and any of the indicators of function. Among participants able to complete a given task, we next used linear regression models to examine self-reported limitations with ADLs and performance on the given test by ApoE carrier status. Similarly, there were no significant relationships between ApoE carrier status and the measures of function. Our estimates provide further confirmation that the ApoE gene may not be a risk factor for functional decline among older Taiwanese adults.     

In silico investigation on the inhibition of Aβ42 aggregation by Aβ40 peptide by potential of mean force study

Recent experimental data revealed that small, soluble Amyloid beta (Aβ₄₂) oligomers, especially dimers impair synaptic plasticity and memory leading to Alzheimer’s disease. Here, we have studied dimerization of Aβ₄₂/Aβ₄₂ homo-dimer and Aβ₄₀/Aβ₄₂ hetero-dimer in terms of free energy profile by all-atom simulations using the ff99SB force field. We have found that in the presence of Aβ₄₀ peptide, there exists a strong tendency to form a hetero-dimer with Aβ₄₂ peptide, suggesting that a possible co-oligomerization. Furthermore, we have investigated the effects of Aβ₄₀ on the Aβ₄₂ peptide. Our study also shows that in presence of Aβ₄₀, the beta-content of Aβ₄₂ monomer is reduced. Additionally, certain residues important for bending in Aβ₄₂ peptide attained an increased flexibility in the presence of Aβ₄₀. The salt-bridge destabilization also manifested the impact of Aβ₄₀ on Aβ₄₂ peptide as a whole. Based on this, one may expect that Aβ₄₀ inhibits the aggregation propensity of Aβ₄₂. Moreover, the binding free energy obtained by the molecular mechanics–Poisson–Boltzmann surface area method also revealed a strong affinity between the two isoforms thereby suggests that Aβ₄₀ binding induces conformational change in Aβ₄₂. Our results suggest that co-oligomerization of Aβ isoforms may play a substantial role in Alzheimer’s disease.     

Construction of a highly thermostable 1,3-1,4-β-glucanase by combinational mutagenesis and its potential application in the brewing industry

Objectives

To improve the thermostability and catalytic property of a mesophilic 1,3-1,4-β-glucanase by combinational mutagenesis and to test its effect in congress mashing.

Results

A mutant β-glucanase (rE-BglTO) constructed by combinational mutagenesis showed a 25 °C increase in optimal temperature (to 70 °C) a 19.5 °C rise in T ₅₀ value and a 15.6 °C increase in melting temperature compared to wild-type enzyme. Its half-life values at 60 and 70 °C were 152 and 99 min, which were 370 and 800 % higher than those of wild-type enzyme. Besides, its specific activity and k _cat value were 42,734 U mg^?1 and 189 s^?1 while its stability under acidic conditions was also improved. In flask fermentation, the catalytic activity of rE-BglTO reached 2381 U ml^?1, which was 63 % higher than that of wild-type enzyme. The addition of rE-BglTO in congress mashing decreased the filtration time and viscosity by 21.3 and 9.6 %, respectively.

Conclusions

The mutant β-glucanase showed high catalytic activity and thermostability which indicated that rE-BglTO is a good candidate for application in the brewing industry.

     

Anatomical evidence for ileal Peyer’s patches innervation by enteric nervous system: a potential route for prion neuroinvasion?

We have examined the innervation of the gut-associated lymphoid system of the sheep ileum, with a view to identifying potential sites for neuroinvasion by pathogens, such as prions (PrP^Sc). Special attention has been paid to the follicles of Peyer’s patches (PPs), which are major sites of PrP^Sc accumulation during infection. Evidence exists that the enteric nervous system, together with the parasympathetic and sympathetic pathways projecting to the intestine, are important for PrP^Sc entry into the central nervous system. Thus, PrP^Sc might move from PPs to the neurons and nerve fibres that innervate them. We investigated, by immunohistochemistry and retrograde tracing (DiI) from the follicles, the distribution and phenotype of enteric neurons innervating the follicles. Antibodies against protein gene product 9.5, tyrosine hydroxylase, dopamine β hydroxylase, choline acetyltransferase, calbindin (CALB), calcitonin gene-related peptide (CGRP), and nitric oxide synthase were used to characterise the neurons. Immunoreactivity for each of these was observed in fibres around and inside PP follicles. CGRP-immunoreactive fibres were mainly seen at the follicular dome. Retrograde tracing revealed submucosal neurons that contributed to the innervation of PPs, including Dogiel type II neurons and neurons immunoreactive for CALB and CGRP. The major source of the adrenergic fibres are the sympathetic ganglia. Our results thus suggest that enteric and sympathetic neurons are involved during the first stage of neuroinvasion, with neurons connecting to them acting as potential carriers of PrP^Sc to the central nervous system. This study was supported by grants from the Ministero dell’Istruzione, dell’Università e della Ricerca (MIUR, PRIN 2006), from the Fondazione del Monte di Bologna e Ravenna and from the National Health and Medical Research Council of Australia (grant no. 400020).     

Triamcinolone solubilization by (2-hydroxypropyl)-β-cyclodextrin: A spectroscopic and computational approach

The molecular foundations of the use of (2-hydroxypropyl)-β-cyclodextrin (HPβCD) as solubility promoter of triamcinolone acetonide (TrA), a corticosteroid with very low aqueous solubility, was investigated by a multidisciplinary spectroscopic and computational approach. Aqueous solutions of TrA and HPβCD were investigated by UV and NMR spectroscopies. The association constant was determined by phase solubility diagrams and by the Foster-Fyfe method whereas the nature of the drug/cyclodextrin aggregates was probed by using the NMR DOSY technique. ROE measurements in solution led to stereochemical information regarding the nature of inclusion processes. TrA/HPβCD powders were prepared and investigated by Raman spectroscopy supported by computational methods. A molecular interaction of the hydroxyacyl chain with cyclodextrin, not identified in solution, was detected. Raman imaging experiments confirmed the attainment of a molecularly homogeneous system when the TrA/HPβCD molar ratio was 1:7 whereas TrA crystallized for mixtures richer in TrA (1:3.5) forming domains with size in the range of 10-15μm. We demonstrate that the combined use of several spectroscopical techniques with specific responsivities allows a detailed depiction of drug/cyclodextrin interaction useful in the development of novel pharmaceutical formulation.     

Considering the potential benefits as well as adverse effects of sun exposure: can all the potential benefits be provided by oral vitamin D supplementation?

Exposure to ultraviolet radiation (UVR) is associated with both adverse and beneficial health effects. While many of the adverse effects of excessive exposure are well known, the adverse effects of insufficient UVR exposure are less clear-cut, but may include a heightened risk of several cancers and autoimmune disorders as well as of bone diseases such as rickets, osteomalacia and osteoporosis. Although some of the postulated beneficial effects of UVR exposure may occur through the maintenance of adequate levels of vitamin D, it is not clear that this can account for all of these effects. We briefly review the epidemiological literature with respect to vitamin D, UVR exposure and autoimmune diseases. We further outline alternative pathways, whereby UVR could alter the risk of development of some cancers and autoimmune disorders, independent of effects on vitamin D synthesis. Recognition of the beneficial effects of UVR exposure has led to a reconsideration of sun avoidance policies. It is important to recognize that all of the beneficial effects of UVR exposure may not occur only through UVR-induced vitamin D synthesis. Thus maintaining current sun avoidance policies while supplementing food with vitamin D may not be sufficient to avoid the risks of insufficient exposure to UVR.     

Probiotics for the skin: a new area of potential application?

AIMS: The current study aimed at assessing, in vitro, the potential use of probiotics for the skin. METHODS AND RESULTS: Propionibacteria were chosen as potential probiotics as they are members of the normal cutaneous microbiota. Dairy strains were chosen because of their documented safe use. Production of anti-microbial substances was assessed, against selected skin pathogens. Only production of organic acids was detected. Two of the tested strains were found to exhibit high adhesion to human keratin, in vitro. Despite this high adhesion, no inhibition of skin pathogen adhesion to human keratin was observed. CONCLUSIONS: The current strains assessed may not be optimal for use as skin probiotics. However, the results of the study show that the methodology works for investigating this kind of application. SIGNIFICANCE AND IMPACT OF THE STUDY: Methods for selecting probiotics for potential application on the skin are presented.     

Elucidating the binding efficacy of β-galactosidase on graphene by docking approach and its potential application in galacto-oligosaccharide production

Herein, we propose the synthesis and characterization of graphene for the immobilization of β-galactosidase for improved galacto-oligosaccharide (GOS) production. The size of synthesized graphene was observed to be 25 nm by TEM analysis while interaction of enzyme with the nanosupport was observed by FTIR spectroscopy. Docking was obtained using molecular docking program Dock v.6.5 while the visual analyses and illustration of protein–ligand complex were investigated by utilizing chimera v.1.6.2 and PyMOL v.1.3 softwares. Immobilized β-galactosidase (IβG) showed improved stability against various physical and chemical denaturants. K _m of IβG was increased to 6.41 mM as compared to 2.38 mM of soluble enzyme without bringing significant change in V _max value. Maximum GOS content also registered an increase in lactose conversion. The maximum GOS production was achieved by immobilized enzyme at specific temperature and time. Hence, the developed nanosupport can be further exploited for developing a biosensor involving β-galactosidase or for immobilization of other industrially/therapeutically important enzymes.     

α-l-Arabinofuranosidases: the potential applications in biotechnology

Recently, alpha-L-arabinofuranosidases (EC3.2.1.55) have received increased attention primarily due to their role in the degradation of lignocelluloses as well as their positive effect on the activity of other enzymes acting on lignocelluloses. As a result, these enzymes are used in many biotechnological applications including wine industry, clarification of fruit juices, digestion enhancement of animal feedstuffs and as a natural improver for bread. Moreover, these enzymes could be used to improve existing technologies and to develop new technologies. The production, mechanisms of action, classification, synergistic role, biochemical properties, substrate specificities, molecular biology and biotechnological applications of these enzymes have been reviewed in this article.     

A highly-active endo-1,3-1,4-β-glucanase from thermophilic Talaromyces emersonii CBS394.64 with application potential in the brewing and feed industries

A 1245-bp endoglucanase gene of glycoside hydrolase (GH) family 7, egl7A, was cloned from the acidothermophilic fungus Talaromyces emersonii CBS394.64 and successfully expressed in Pichia pastoris. Sequence alignments indicated that Egl7A had highest identity of 62.7% at the amino acid level with the functionally characterized endoglucanase from Aspergillus terreus NIH2624. Purified recombinant Egl7A exhibited the maximum activity at pH 4.5 and 70 °C, retained stable over the pH range of 2.0–12.0 and at 65 °C, and was strongly resistant to acidic and neutral proteases, most metal ions and SDS. The enzyme exhibited the highest specific activity reported so far (11,299 U mg⁻¹) when using barley β-glucan as the substrate. Egl7A exhibited broad substrate specificity, including barley β-glucan, lichenin, CMC-Na, and xylan and had capacity to cleave cellopentaose and cellohexaose into smaller units rapidly. Under simulated mashing conditions, addition of Egl7A reduced the mash viscosity by 12.40%; when combined with a GH10 xylanase, more viscosity reduction (27.75%) was observed, which is significantly higher than that of the commercial enzyme Ultraflo XL (17.91%). All these properties make Egl7A attractive for potential applications in the feed and brewing industries.     

Hydroxymethylnitrofurazone:Dimethyl-β-cyclodextrin Inclusion Complex: A Physical–Chemistry Characterization

Hydroxymethylnitrofurazone (NFOH) is active against Trypanosoma cruzi; however, its low solubility and high toxicity precludes its current use in treatment of parasitosis. Cyclodextrin can be used as a drug carrier system, as it is able to form inclusion (host–guest) complexes with a wide variety of organic (guest) molecules. Several reports have shown the interesting use of modified β-cyclodextrins in pharmaceutical formulation, to improve the bioavailability of drugs and to decrease their toxicity. The aim of this work was to characterize inclusion complexes formed between NFOH and dimethyl-β-cyclodextrin (DM-β-CD) by complexation/release kinetics and solubility isotherm experiments using ultraviolet (UV)-visible spectrophotometry and by the measurement of the dynamics information obtained from T ₁ relaxation times and diffusion (DOSY) experiments using nuclear magnetic resonance (NMR) spectroscopy. The complex was prepared at different NFOH and DM-β-CD molar ratios. The UV-visible measurements were recorded in a spectrophotometer, and NMR experiments were recorded at 20°C on a NMR spectrometer (Varian Inova) operating at 500 MHz. Longitudinal relaxation times were obtained by the conventional inversion-recovery method and the DOSY experiments were carried out using the BPPSTE sequence. The kinetics of complexation revealed that 30 h is enough for stabilization of the NFOH absorbance in presence of cyclodextrin. Solubility isotherm studies show a favorable complexation and increase in solubility when NFOH interacts with cyclodextrin. The analysis of the NMR-derived diffusion coefficients and T ₁ relaxation times shows that in the presence of DM-β-CD, NFOH decreases its mobility in solution, indicating that this antichagasic compound interacts with the cyclodextrin cavity. The release kinetics assays showed that NFOH changes its release profile when in the presence of cyclodextrin due to complexation. This study was focused on the physicochemical characterization of drug-delivery formulations that may serve as potentially new therapeutic options for the treatment of Chagas’ disease.     

Do oral bacteria alter the regenerative potential of stem cells? A?concise review

Mesenchymal stem cells (MSCs) are widely recognized as critical players in tissue regeneration. New insights into stem cell biology provide evidence that MSCs may also contribute to host defence and inflammation. In case of tissue injury or inflammatory diseases, e.g. periodontitis, stem cells are mobilized towards the site of damage, thus coming in close proximity to bacteria and bacterial components. Specifically, in the oral cavity, complex ecosystems of commensal bacteria live in a mutually beneficial state with the host. However, the formation of polymicrobial biofilm communities with pathogenic properties may trigger an inadequate host inflammatory‐immune response, leading to the disruption of tissue homoeostasis and development of disease. Because of their unique characteristics, MSCs are suggested as crucial regulators of tissue regeneration even under such harsh environmental conditions. The heterogeneous effects of bacteria on MSCs across studies imply the complexity underlying the interactions between stem cells and bacteria. Hence, a better understanding of stem cell behaviour at sites of inflammation appears to be a key strategy in developing new approaches for in situ tissue regeneration. Here, we review the literature on the effects of oral bacteria on cell proliferation, differentiation capacity and immunomodulation of dental‐derived MSCs.     

Sex differences in cannabinoid pharmacology: A reflection of differences in the endocannabinoid system?

Marijuana is the most widely used illicit drug in the U.S., and marijuana use by women is on the rise. Women have been found to be more susceptible to the development of cannabinoid abuse and dependence, have more severe withdrawal symptoms, and are more likely to relapse than men. The majority of research in humans suggests that women are more likely to be affected by cannabinoids than men, with reports of enhanced and decreased performance on various tasks. In rodents, females are more sensitive than males to effects of cannabinoids on tests of antinociception, motor activity, and reinforcing efficacy. Studies on effects of cannabinoid exposure during adolescence in both humans and rodents suggest that female adolescents are more likely than male adolescents to be deleteriously affected by cannabinoids. Sex differences in response to cannabinoids appear to be due to activational and perhaps organizational effects of gonadal hormones, with estradiol identified as the hormone that contributes most to the sexually dimorphic effects of cannabinoids in adults. Many, but not all sexually dimorphic effects of exogenous cannabinoids can be attributed to a sexually dimorphic endocannabinoid system in rodents, although the same has not yet been established firmly for humans. A greater understanding of the mechanisms underlying sexually dimorphic effects of cannabinoids will facilitate development of sex-specific approaches to treat marijuana dependence and to use cannabinoid-based medications therapeutically.     

Quantum mechanical study of the inclusion process of adamantanol isomers by l-tryptophan-modified-β-cyclodextrin

The complexation processes of 1 and 2-adamantanol with l-tryptophan-β-cyclodextrin have been studied using ab initio Hartree–Fock and density functional theory levels. For the host: guest inclusion processes, the up mode with the OH group of the alcohol oriented towards the secondary rim, is found to be in qualitative agreement with the experimental finding. A molecular recognition mechanism is proposed based on the host: guest relative dipole orientation. For the complex with the 2-Ada isomer the host and guest the dipoles are parallels favoring the interaction energy and. This mechanism can explain the small energy difference for the processes involving the adamantanol isomers and modified cyclodextrins.     

Gero Miesenböck: instructing the nervous system. Interview by William A Wells

Gero Miesenböck uses light and genetically encoded sensors and actuators to observe and control neural activity. Having caused headless flies to fly at will, he is set to understand how the nervous system encodes behavior.New techniques often drive advances in cell biology. Gero Miesenböck has developed powerful tools that open up whole new regions of neurobiology.First came tools to measure neuronal activity. The synaptolucins (1), developed with Jim Rothman (Memorial Sloan-Kettering Cancer Center, New York, NY), were based on a luciferase fusion protein targeted to synaptic vesicles. Neuronal activity led to exocytosis, thus exposing the synaptolucin to an extracellular pool of its substrate luciferin. Later came a pH-sensitive GFP (pHluorin) that lit up when exocytosis propelled it from the acidic inside of the secretory vesicle to the neutral extracellular environment (2).Miesenböck started his own lab in 1999 and is currently at Yale University (New Haven, CT). His chARGe system turned light into neural activity: it co-opted a fly visual transduction pathway to detect light and convert it into a depolarizing signal in neurons (3). In a simpler phototrigger system, light removed the chemical protection from ligands, allowing them to bind and activate receptors that could be expressed in any neuron subset of interest (4). He used this latter system to get the headless flies flying. In contrast to direct electrical stimulation of neurons using electrodes, the phototrigger can control neuron subtypes that are dispersed amongst other cells, all the while leaving animals free to roam and behave.Open in a separate windowFigure 1A light-activated circuit gets a sluggish fly (left) moving (right).MIESENBÖCK/ELSEVIERMiesenböck has recorded the first images of information flow between specific classes of neurons in an intact brain (5), and he recently discovered a new kind of circuit within the olfactory system (6). This circuit spreads excitation laterally, perhaps helping to boost otherwise sub-threshold signals so they can be detected (6). We spoke to Miesenböck as he was preparing to move to the University of Oxford, UK, where he will be helping to build up neuroscience in the Department of Physiology.