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Vesicle fluctuation analysis of the effects of sterols on membrane bending rigidity.

Sterols are regulators of both biological function and structure. The role of cholesterol in promoting the structural and mechanical stability of membranes is widely recognized. Knowledge of how the related sterols, lanosterol and ergosterol, affect membrane mechanical properties is sparse. This paper presents a comprehensive comparison of the effects of cholesterol, lanosterol, and ergosterol upon the bending elastic properties of 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine giant unilamellar vesicles. Measurements are made using vesicle fluctuation analysis, a nonintrusive technique that we have recently improved for determining membrane bending rigidity. Giving a detailed account of the vesicle fluctuation analysis technique, we describe how the gravitational stabilization of the vesicles enhances image contrast, vesicle yield, and the quality of data. Implications of gravity on vesicle behaviour are also discussed. These recent modifications render vesicle fluctuation analysis an efficient and accurate method for determining how cholesterol, lanosterol, and ergosterol increase membrane bending rigidity.

Computer Simulation↗

Differential toxicity of camptothecin, topotecan and 9-aminocamptothecin to human, canine, and murine myeloid progenitors (CFU-GM) in vitro.

PURPOSE: 20(S)-Camptothecin (CAM), topotecan (TPT, active ingredient in Hycamtin) and 9-amino-20(S)-camptothecin (9AC) are topoisomerase I inhibitors that cause similar dose-limiting toxicities to rapidly renewing tissues, such as hematopoietic tissues, in humans, mice, and dogs. However, dose-limiting toxicity occurs at tenfold lower doses in humans than in mice. The purpose of the current study was to determine whether hematopoietic progenitors of the myeloid lineage from humans, mice, and dogs exhibit the differential sensitivity to these compounds that is evident in vivo. METHODS: Drug-induced inhibition of in vitro colony formation by a myeloid progenitor in human, murine, and canine marrow colony-forming unit-granulocyte/macrophage (CFU-GM) provided the basis for interspecies comparisons at concentrations which inhibited colony formation by 50% (IC50) and 90% (IC90). RESULTS: Murine IC90 values were 2.6-, 2.3-, 10-, 21-, 5.9-, and 11-fold higher than human values for CAM lactone (NSC-94600) and sodium salt (NSC-100880), TPT (NSC-609699), and racemic (NSC-629971), semisynthetic and synthetic preparations (NSC-603071) of 9AC, respectively. In contrast, canine IC90 values were the same as, or lower than, the human IC90 values for all six compounds. CONCLUSIONS: The greater susceptibility of humans and dogs to the myelotoxicity of camptothecins, compared to mice, was evident in vitro at the cellular level. Differential sensitivity between murine and human myeloid progenitors explains why the curative doses of TPT and 9AC in mice with human tumor xenografts are not achievable in patients. Realizing the curative potential of these compounds in humans will require the development of therapies to increase drug tolerance of human CFU-GM at least to a level equal to that of murine CFU-GM. Because these interspecies differences are complicated by species-specific effects of plasma proteins on drug stability, not all in vitro assay conditions will yield results which can contribute to the development of such therapies.

Animals↗

Isolation by genetic and physiological characteristics of a fuel-ethanol fermentative Saccharomyces cerevisiae strain with potential for genetic manipulation.

Fuel ethanol fermentation process is a complex environment with an intensive succession of yeast strains. The population stability depends on the use of a well-adapted strain that can fit to a particular industrial plant. This stability helps to keep high level of ethanol yield and it is absolutely required when intending to use recombinant strains. Yeast strains have been previously isolated from different distilleries in Northeast Brazil and clustered in genetic strains by PCR-fingerprinting. In this report we present the isolation and selection of a novel Saccharomyces cerevisiae strain by its high dominance in the yeast population. The new strain, JP1 strain, presented practically the same fermentative capacity and stress tolerance like the most used commercial strains, with advantages of being highly adapted to different industrial units in Northeast Brazil that used sugar cane juice as substrate. Moreover, it presented higher transformation efficiency that pointed out its potential for genetic manipulations. The importance of this strain selection programme for ethanol production is discussed.

Acids↗

Labile oligomeric structure of human placental 15-hydroxyprostaglandin dehydrogenase.

NAD-dependent 15-hydroxyprostaglandin dehydrogenase has been isolated from human term placenta. About 9,000-fold enrichment was achieved with a yield of 7.6%. Electrophoretic analyses suggested that glycerol stabilized an active structure of the enzyme, and sodium dodecyl sulfate might dissociate it. The instability of the enzyme activity may relate to its labile oligomeric structure which is easily dissociated into subunits.

Electrophoresis, Polyacrylamide Gel↗

Sensory gating in normals and schizophrenics: a failure to find strong P50 suppression in normals.

In a series of investigations, suppression of the auditory-evoked P50 potential to the second of two paired clicks presented 500 msec apart has been shown to be absent in schizophrenic patients, whereas normals suppress their second response to less than 20% of the first response. The phenomenon has been discussed as a possible trait marker for schizophrenia. The present study with 19 schizophrenics and 23 healthy control subjects was intended as an extended replication of the phenomenon using different stimulus parameters and a slightly different method of measuring P50 amplitudes. Replication was unsuccessful, revealing only weak suppression scores in normal subjects not significantly superior to schizophrenics. Retest sessions yielded generally stronger P50 suppression suggesting that the stability of the measure over time is questionable. The methodological changes are discussed as possible sources of this failure to replicate. It is concluded that the conditions under which P50-suppression occurs should be better clarified in order to facilitate replication.

Acoustic Stimulation↗

Chloride attachment negative-ion mass spectra of sugars by combined liquid chromatography and atmospheric pressure chemical ionization mass spectrometry.

A method has been developed for the rapid determination of sugars, including molecular weight measurements, using high-performance liquid chromatography coupled with negative-ion, atmospheric-pressure chemical-ionization mass spectrometry. The chromatography was carried out on a 250 x 4 mm I.D. column packed with 7 microns NH2-silica. The mobile phase consisted of a high percentage of methanol or acetonitrile with a small amount of chloroform. During the mass spectrometry, a strong base is formed from the polar solvent molecules by corona discharge, followed by ion-molecule reactions in the chemical ionization ion source (e.g. the methoxy anion is formed from methanol). This strong base reacts with the chloroform, generating chloride ions, which in turn react with the neutral sugar molecules as they elute from the chromatograph. The chloride ion and sugar interactions yield chloride-attachment ions, which are further stabilized by successive collisions. In this method, authentic monosaccharides and some oligosaccharides show dominant quasi-molecular ions, [M + Cl]-, with little fragmentation, and its particularly useful for the molecular weight determination of sugars.

Atmospheric Pressure↗

Relocating expression of vegetative insecticidal protein into mother cell of Bacillus thuringiensis.

Vegetative insecticidal protein (VIP) is a class of insecticidal proteins produced by some strains of Bacillus thuringiensis during the vegetative stage of their growth. Unlike delta-endotoxins which are produced as parasporal inclusion bodies within the cell during sporulation, VIP is secreted into the culture medium. Here we report the relocation of the expression of VIP into the mother cell compartment in a manner similar to well-characterized Cry proteins. Relocation of VIP is directed to mother cell by placing its synthesis under sporulation-dependent promoters, BtI and BtII. The insertion of cry preferred transcription termination sequence at the 3(') region and a STAB-SD sequence at the 5(') region of the gene provided stability to the vip transcript and enhanced its yield. The demonstrated expression of VIP within the cells in the form of inclusion bodies would facilitate development of a suitable formulation for the application of this class of insecticidal proteins in the field.

Bacillus thuringiensis↗

Disentangling the contribution of the paretic and non-paretic ankle to balance control in stroke patients.

During stroke recovery, restoration of the paretic ankle and compensation in the non-paretic ankle may contribute to improved balance maintenance. We examine a new approach to disentangle these recovery mechanisms by objectively quantifying the contribution of each ankle to balance maintenance. Eight chronic hemiparetic patients were included. Balance responses were elicited by continuous random platform movements. We measured body sway and ground reaction forces below each foot to calculate corrective ankle torques in each leg. These measurements yielded the Frequency Response Function (FRF) of the stabilizing mechanisms, which expresses the amount and timing of the generated corrective torque in response to sway at the specified frequencies. The FRFs were used to calculate the relative contribution of the paretic and non-paretic ankle to the total amount of generated corrective torque to correct sway. All patients showed a clear asymmetry in the balance contribution in favor of the non-paretic ankle. Paretic balance contribution was significantly smaller than the contribution of the paretic leg to weight bearing, and did not show a clear relation with the contribution to weight bearing. In contrast, a group of healthy subjects instructed to distribute their weight asymmetrically showed a one-on-one relation between the contribution to weight bearing and to balance. We conclude that the presented approach objectively quantifies the contribution of each ankle to balance maintenance. Application of this method in longitudinal surveys of balance rehabilitation makes it possible to disentangle the different recovery mechanisms. Such insights will be critical for the development and evaluation of rehabilitation strategies.

Adult↗

Morphology-engineered NiFe@C nanocages boosting electrochemical quantification of ractopamine in meat samples.

It is essential to acquire efficient electrocatalysts to develop ractopamine (RAC) electrochemical sensors. Herein, we report the synthesis of a series of carbon coated NiFe alloy nanostructures (e.g., NiFe@C nanoparticles, nanocubes and nanocages) using NiFe Prussian blue analogue (PBA) as the precursor. The NiFe@C nanocages exhibited the best electrocatalytic performance for RAC sensing. This is attributed to the embedded NiFe alloy nanoparticles that provide abundant active sites, and the unique nanocage structure facilitates electron transfer pathways while offering a high specific surface area. The resulting sensor achieves a low detection limit (LOD) of 54 nM (S/N = 3) within a linear range of 0.2-12 μM. Moreover, the sensor demonstrates good reproducibility, stability, and excellent long-term stability. Practical applicability was confirmed in meat samples, yielding satisfactory recovery rates ranging from 98% to 108%. A feasible strategy was introduced herein for rational design of metal@carbon electrocatalysts.

Phenethylamines↗

Phenotypes and fed-batch fermentation of ubiquinone-overproducing fission yeast using ppt1 gene.

Ubiquinone (UQ), a component of the electron transfer system in many organisms, has been widely used for pharmaceuticals and cosmetics. In this study, we cloned and overexpressed the full-length ppt1 (MTppt1) gene, which encodes p-hydroxybenzoate:polyprenyltransferase and ERppt1 gene, which was modified to be localized on endoplasmic reticulum in fission yeast. The yeast MTppt1 and ERppt1 transgenic lines showed about 3.7 and 5.1 times increment in UQ content and the recombinant yeasts with a higher UQ level are more resistant to H(2)O(2), Cu(2+) and NaCl, and interestingly their growth was also faster than the wild type at lower temperature. For large-scale cultivation, the direct feedback control of glucose using an on-line ethanol concentration monitor for ubiquinone production of yeast ERppt1 by high-cell-density fermentation was investigated and the fermentation parameters (e.g., dissolved oxygen, pH, ethanol concentration, oxygen uptake rate, carbon dioxide evolution rate and respiration quotient) were also discussed. After 90 h cultures, the yeast dry cell weight reached 57 gl(-1) and the ubiquinone yield reached 23 mgl(-1). In addition, plasmid stability was maintained at high level throughout the fermentation.

Biomass↗

Adsorption of citric acid from dilute aqueous solutions by hydroxyapatite.

The role of citric acid in the demineralization of dental enamel, which is mainly constituted by hydroxyapatite, is important for periodontal regeneration and in the conditioning of enamel or dentin for bonding restorative resins. The adsorption of citric acid from aqueous solutions onto synthetic hydroxyapatite at 278, 288, 298, and 308 K and pH 4.8 has been studied by means of UV spectroscopy. The adsorption reaction, which takes place by an interaction between phosphate groups and citrate anions at the solid-solution interface, yields an adsorbate-adsorbent complex of high stability. The adsorption isotherms fit the Langmuirian shape. The proposed adsorption model, where citrate species interact in a bidentate manner (one citrate ion links two phosphate sites), is coherent with the experimental data. The activation standard heat and activation standard entropy were calculated. All the thermodynamic and kinetic parameters were in concordance with the adsorption reaction proposed in this work.

Journal Article↗

Solubilization and characterization of erythropoietin receptor from transplantable mouse erythroblastic leukemic cells.

Functional erythropoietin (Ep) receptor (Ep-R) on transplantable mouse leukemic cells was solubilized by 3[(3-cholamidopropyl)dimethyl ammonio]-1-propanesulfate (CHAPS). An assay of the solubilized Ep-R was established: (1) radioiodinated Ep was bound to the solubilized Ep-R, (2) Ep.Ep-R complexes were covalently cross-linked, (3) the cross-linked products were selectively precipitated by polyethylene glycol, (4) the products were separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and (5) the Ep.Ep-R cross-linked products were measured. Optimal conditions to yield the cross-linked products and to stabilize the solubilized Ep-R were found. The solubilized Ep-R had properties similar to those of Ep-R on the membrane with respect to binding with Ep and the cross-linked products. These results would be exploited to allow purification of Ep-R.

Animals↗

Chemical formation of 4-hydroxy-2,5-dimethyl-3[2H]-furanone from D-fructose 1,6-diphosphate.

The selective chemical formation of 4-hydroxy-2,5-dimethyl-3[2H]-furanone (HDF) from D-fructose 1,6-diphosphate in the presence of reduced nicotinamide-adenine-dinucleotides (NAD(P)H) was investigated by means of HPLC-DAD and HPLC-UV-MS/MS. The temperature optimum for HDF formation was 30 degrees C, whereas the pH value (pH 3-10) and chemical nature of the buffer had no significant influence. A linear correlation of reaction time and D-fructose 1,6-diphosphate concentration with the obtained HDF yield was observed. Proteins appeared to have a stabilizing effect. The NAD(P)H were mandatory, even in the presence of protein, implying a non-enzymatic hydride-transfer to an unknown intermediate which finally leads to the selective formation of HDF. The hydride-transfer was confirmed by the application of selectively pro-4R or pro-4S deuterium labeled NADH resulting in each case in the formation of HDF exhibiting a deuterium labeling of approx 30% and employment of [4R,S-(2)H(2)]-NADH led to a deuterium labeling of approx 66%. The incubation of [1-(13)C]-D-fructose 1,6-diphosphate with [4R,S-(2)H(2)]-NADH revealed that the hydride is transferred to C-5 or C-6 of the D-fructose 1,6-diphosphate skeleton. Thus, a chemical HDF formation from D-fructose 1,6-diphosphate under physiological reaction conditions was shown and for the first time to our knowledge a non-enzymatic hydride-transfer from NADH to a carbohydrate structure was demonstrated.

Deuterium↗

Conversion of p-methoxyphenyl glycosides into the corresponding glycosyl chlorides and bromides, and into thiophenyl glycosides.

p-Methoxyphenyl (pMP) beta-D-glycopyranosides (Glc, Gal, GlcNPhth, GalNPhth, GlcNTroc, Gal beta 4Glc, Gal alpha 4Gal) were prepared from the corresponding 1-O-acetyl sugars in 79-90% yield, using boron trifluoride etherate as promoter. Treatment of the pMP glycosides with acyl chlorides or bromides in the presence of various Lewis acids gave the corresponding glycosyl chlorides and bromides in 81-98% yield. Treatment of the acyl-protected pMP glycosides with thiophenol and boron trifluoride etherate gave the corresponding thioglycosides in 80-100% yield and high (> 20:1) beta/alpha selectivity. The stability of pMP glycosides was investigated against a series of reagents.

Anisoles↗

New method for the preparation of highly stable polysiloxane-coated glass open-tubular capillary columns and application to the analysis of hormonal steroids.

A new approach to the preparation of methylsiloxane-coated glass open-tubular capillary columns is described. A siloxane polymeric mixture is first prepared by hydrolysis of dimethyldichlorosilane. The polymer is then coated on to the previously etched glass wall by a base-catalyzed reaction to yield a non-polar system of high stability and showing satisfactory chromatographic properties. This type of column has been used for the high-resolution separations of complex hormonal steroid mixtures. The flexibility of this method should permit the preparation of capillary systems with a wide range of polarity.

Adult↗

Reproduction numbers and sub-threshold endemic equilibria for compartmental models of disease transmission.

A precise definition of the basic reproduction number, R0, is presented for a general compartmental disease transmission model based on a system of ordinary differential equations. It is shown that, if R0<1, then the disease free equilibrium is locally asymptotically stable; whereas if R0>1, then it is unstable. Thus, R0 is a threshold parameter for the model. An analysis of the local centre manifold yields a simple criterion for the existence and stability of super- and sub-threshold endemic equilibria for R0 near one. This criterion, together with the definition of R0, is illustrated by treatment, multigroup, staged progression, multistrain and vector-host models and can be applied to more complex models. The results are significant for disease control.

Animals↗

Personality structure in patients with winter depression, assessed in a depression-free state according to the five-factor model of personality.

BACKGROUND: Depressive disorders are often associated with abnormalities of personality. This has also been found in Seasonal Affective Disorder, Winter Depression type (W-SAD), although relatively few and small studies have been done on this disorder so far, and mainly in the depressed phase. The aim of this study was to explore the personality structure and its possible background and clinical correlates in a larger group of patients with W-SAD, assessed in a non-depressed state, in the Five-Factor (or "Big Five") model of personality paradigm. METHODS: 82 persons (87% of those asked) with diagnosed W-SAD, most of them originally self-referred, who had previously received light treatment at our unit, completed a Norwegian version of an inventory yielding the personality factors Agreeableness, Surgency, Conscientiousness, Emotional stability, and Openness to experience. RESULTS: Compared to a Norwegian reference group, the mean scores of the W-SAD patients were significantly lower on the factors agreeableness, surgency and emotional stability, and significantly higher on conscientiousness. However, very few patients had scores outside the reference range. The personality factors do not seem to be associated with the core physiological manifestations of winter depression, but rather with the psychic aspects, and are poor predictors of response to light treatment. LIMITATIONS: Some reservations should be made as to the generalisability of the findings to SAD patients in general. Thus, increased conscientiousness may reflect help-seeking behaviour. CONCLUSIONS: Personality structure does not seem to predict occurrence of physiological reactions to lack of adequate daylight in autumn/winter, and is a poor predictor of improvement after light treatment, but may predict the associated psychic symptomatology.

Adult↗

Avidin-biotin immobilization of unilamellar liposomes in gel beads for chromatographic analysis of drug-membrane partitioning.

To construct a homogeneous lipid membrane chromatographic phase, biotinylated unilamellar liposomes of small and large sizes (SUVs and LUVs, respectively) were immobilized in avidin- or streptavidin-derived gel beads in amounts up to 55 micromol phospholipid/ml gel bed at yields above 50%. The immobilized liposomes exhibited excellent stability due to avidin-biotin multiple-site binding. The trapped volume and size distribution of the immobilized liposomes (0.33-0.42 microl/micromol lipid and 20-30 nm diameter for SUVs, 1.7-1.9 microl/micromol lipid and 80-120 nm for LUVs) indicated the unilamellarity and integrity of the immobilized liposomes. Partitioning of 15 pharmaceutical drugs into the bilayers of LUVs immobilized in different gel matrices correlated very well, as shown by chromatographic drug retention analysis. The partitioning of several beta-blockers into the immobilized LUVs showed a close correlation with their partitioning, reported in the literature, into free liposomes. The avidin-biotin-immobilized unilamellar liposomes can thus be used for chromatographic analysis and screening of solute-membrane interactions.

Adrenergic beta-Antagonists↗