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PubMed2006

An AFM investigation of oligonucleotides anchored on unoxidized crystalline silicon surfaces.

Carboxylic terminated monolayers have been covalently attached on phosphorous doped crystalline (100) silicon surfaces using a cathodic electro grafting technique. The functionalization concentration and efficiency have been evaluated with different techniques. In particular, topographic images, performed with an atomic force microscope, were used to optimize the protocol in order to obtain a surface whose characteristics of uniformity and reproducibility are ideal for a bio-electronic device. Phase lag images of the functionalized surfaces were also performed, and show non-topographic structures that have been interpreted as areas of different molecule self-orientation. Poly-thymine oligonucleotides have been anchored on such a surface to form a nano-biosensing device capable to react selectively with a specific target molecule, a poly-adenine oligonucleotide. AFM images of high density (approximately 3x10(12) mol/cm2) single strand and double strand covered samples show toroidal shaped structures formed by the self-assembly of the oligonucleotides on the silicon surface.

Coated Materials, Biocompatible↗
PubMed2004

A new application of stopped-flow chiral HPLC: inversion of enantiomer elution order.

A newly developed procedure to reverse the enantiomer elution order of compounds resolved on chiral stationary phases (CSPs) for HPLC is presented. The optimized analytical protocol is based on the effect of temperature on enantioselectivity and does not involve any changing in mobile phase composition or type of CSP. In essence, the approach entails variable temperature chromatography at two temperatures. The enantiomer separation is performed at a low column temperature, with stopping the flow prior to elution of the less retained enantiomer. Then, the column temperature is changed with the peaks trapped inside the column, followed by elution with the same mobile phase in reverse direction. Under these conditions, the more pronounced loss in free energy of binding for the more strongly bound enantiomer results in an inversion of the elution order. This procedure may be applied to each enantiomer pair that is separated by chiral HPLC under an appreciable enthalpy-control.

Chromatography, High Pressure Liquid↗
PubMed2006

Proteins modification of poly(dimethylsiloxane) microfluidic channels for the enhanced microchip electrophoresis.

This report described proteins modification of poly(dimethylsiloxane) (PDMS) microfluidic chip based on layer-by-layer (LBL) assembly technique for enhancing separation efficiency. Two kinds of protein-coated films were prepared. One was obtained by successively immobilizing the cationic polyelectrolyte (chitosan, Chit), gold nanoparticles (GNPs), and protein (albumin, Albu) to the PDMS microfluidic channels surface. The other was achieved by sequentially coating lysozyme (Lys) and Albu. Neurotransmitters (dopamine, DA; epinephrine, EP) and environmental pollutants (p-phenylenediamine, p-PDA; 4-aminophenol, 4-AP; hydroquinone, HQ) as two groups of separation models were studied to evaluate the effect of the functional PDMS microfluidic chips. The results clearly showed these analytes were efficiently separated within 140 s in a 3.7 cm long separation channel and successfully detected with in-channel amperometric detection mode. Experimental parameters in two protocols were optimized in detail. The detection limits of DA, EP, p-PDA, 4-AP, and HQ were 2.0, 4.7, 8.1, 12.3, and 14.8 microM (S/N=3) on the Chit-GNPs-Albu coated PDMS/PDMS microchip, and 1.2, 2.7, 7.2, 9.8, and 12.2 microM (S/N=3) on the Lys-Albu coated one, respectively. In addition, through modification, the more homogenous channel surface displayed higher reproducibility and better stability.

Buffers↗
PubMed2006

Efficient and non-denaturing membrane solubilization combined with enrichment of membrane protein complexes by detergent/polymer aqueous two-phase partitioning for proteome analysis.

It is of central interest in membrane proteomics to establish methods that combine efficient solubilization with enrichment of proteins and intact protein complexes. We have investigated the quantitative and qualitative solubilization efficiency of five commercially available detergents using mitochondria from the yeast Saccharomyces cerevisiae as model system. Combining the zwitterionic detergent Zwittergent 3-10 and the non-ionic detergent Triton X-114 resulted in a complementary solubilization of proteins, which was similar to that of the anionic detergent sodium dodecyl sulfate (SDS). The subsequent removal of soluble proteins by detergent/polymer two-phase system partitioning was further enhanced by addition of SDS and increasing pH. A large number of both integral and peripheral membrane protein subunits from mitochondrial membrane protein complexes were identified in the detergent phase. We suggest that the optimized solubilization protocol in combination with detergent/polymer two-phase partitioning is a mild and efficient method for initial enrichment of membrane proteins and membrane protein complexes in proteomic studies.

Cell Membrane↗
PubMed2005

Misoprostol alone for early abortion: an evaluation of seven potential regimens.

INTRODUCTION: A growing body of literature has shown that misoprostol alone could be effective for early medical abortion. We evaluated seven potential regimens in women up to 56 days of gestation in order to potentially identify an optimal regimen. METHODS: In phase I of the study, women requesting early abortion were randomized to one of three misoprostol regimens (4x400 microg po every 3 h, 2x800 microg po every 6 h, 1x600 pv microg); in phase II, women were randomized to one of two regimens (2x800 microg po every 3 h, 1x800 pv microg). In phase III, we consecutively tested two regimens (800 microg pv wetted with saline repeated after 24 h if intact gestational sac, 2x800 microg pv wetted with saline) to validate previously published results. RESULTS: Although most women experienced some side effects, all regimens were tolerable and acceptable. Five of the seven regimens resulted in complete abortion rates of 60% or less. Only repeated doses of 800 microg pv misoprostol resulted in efficacy exceeding 60%. DISCUSSION: Misoprostol-alone abortion regimens using oral misoprostol are too ineffective for clinical use or further investigation. Regimens with repeated dosing of misoprostol 800 microg pv warrant further study to find the optimal treatment protocol.

Abdominal Pain↗
PubMed2006

Sperm viability in the black-footed ferret (Mustela nigripes) is influenced by seminal and medium osmolality.

Fundamental knowledge of spermatozoa cryobiology can assist with optimizing cryopreservation protocols needed for genetic management of the endangered black-footed ferret. Objectives were to characterize semen osmolality and assess the influence of two media at various osmolalities on sperm viability. We examined the influence of Ham's F10 +Hepes medium (H) at 270, 400, 500 or 700 mOsm (adjusted with sucrose, a nonpermeating cryoprotectant) and TEST Yolk Buffer (TYB) with 0% (300 mOsm) versus 4% (900 mOsm) glycerol (a permeating cryoprotectant). Electroejaculates (n=16) were assessed for osmolality using a vapor pressure osmometer. For media comparison, semen (n=5) was collected in TYB 0%, split into six aliquots, and diluted in H270, H400, H500, H700, and TYB 0% or TYB 4%. Each sample was centrifuged (300 g, 8 min), resuspended in respective medium, and maintained at 37 degrees C for 3h. Sperm motility and forward progression were monitored every 30 min for 3h post-washing. Acrosomal integrity was monitored at 0 and 60 min post-washing. Results demonstrated that black-footed ferret semen has a comparatively high osmolality (mean+/-SEM, 513.1+/-32.6 mOsm; range, 366-791 mOsm). Ferret spermatozoa were sensitive to hyperosmotic stress. Specifically, sperm motility was more susceptible (P<0.01) to hyperosmotic conditions than acrosomal integrity, and neither were influenced (P>0.05) by hypotonic solutions. Exposure to TYB 4% glycerol retained more (P<0.01) sperm motility than a hyperosmotic Ham's (700 mOsm). These findings will guide the eventual development of assisted breeding with cryopreserved sperm contributing to genetic management of this rare species.

Acrosome↗
PubMed2006

Use of "2-dose" regimen of methotrexate to treat ectopic pregnancy.

OBJECTIVE: To evaluate the safety and acceptability of a novel dosing regimen of methotrexate to treat ectopic pregnancy. DESIGN: Prospective study. SETTING: Three academic medical centers. PATIENT(S): One hundred one patients with ectopic pregnancy who elected to have medical therapy. INTERVENTION(S): Intramuscular methotrexate 50 mg/m(2) was administered on days 0 and 4; additional doses of methotrexate were given on day 7 and/or day 11 if hCG levels did not decrease by 15% during the follow-up period. MAIN OUTCOME MEASURE(S): Adverse events, acceptability, and resolution of pregnancy without surgical treatment. RESULT(S): With this protocol, 87% of patients were treated successfully. Of those protocols that were considered failures, only 3% of patients experienced rupture of ectopic pregnancy. Treatment was well-tolerated; most side effects were reported as mild and transient. Ninety-one percent of the 61.5% of patients who responded to the satisfaction questionnaire reported satisfaction with this regimen. CONCLUSION(S): This "2-dose" protocol minimizes the number of injections and surveillance visits, compared with the "multiple dose" regimen, and methotrexate is administered more frequently than with the "single dose" regimen. The protocol may optimize the balance between convenience and efficacy. In a limited number of women, no safety concerns were noted with up to 4 doses of methotrexate in a 2-week period without leucovorin rescue.

Adult↗
PubMed2005

[Cryopreservation of immature testicular tissue].

An increased incidence of cancer is observed in the population of adolescents and young adults since thirty years. Major progress in cancer diagnosis and therapy is unfortunately associated to high degree of toxicity on gonad function. Cryopreservation of ovarian tissue is performed in girls and women before cancer treatment with high risk of infertility. Procedures for ejaculated or testicular extracted spermatozoa are well defined. However, for prepubertal boys or after ejaculated sperm collection failure, mature or immature testicular tissue banking should be proposed. Still, an optimal cryopreservation protocol is a prerequisite for clinical application and does not exist for the moment. Furthermore, the future applications of immature testicular tissue banking should be developed, not solely germ cell in vitro maturation but also autologous testicular tissue grafting.

Animals↗
PubMed2004

MALDI-TOF-MS analysis of bacterial spores: wet heat-treatment as a new releasing technique for biomarkers and the influence of different experimental parameters and microbiological handling.

Short wet heat-treatment is presented as a new technique to release high-mass biomarkers to obtain strain-specific fingerprints of intact bacterial spores by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS). Wet heat-treatment was applied for several minutes (3-30) by two techniques using either a screw-cap tube submerged in a glycerol bath at 120 degrees C or an Eppendorff-tube submerged in a water bath at 100 degrees C. Both techniques turned out to be successful for releasing high-mass biomarkers. The influence of different experimental parameters and microbiological handling on the peak pattern of the released high-mass biomarkers was studied. While the sporulation medium, the applied washing procedure, and the choice of matrix crucially influenced the peak pattern, other parameters like storage conditions were found to be insignificant. A protocol of optimized experimental conditions for MALDI-MS of wet heat-treated spores is presented.

Bacillus↗
PubMed2004

Optimized suppression of adducts in polymerase chain reaction products for semi-quantitative SNP genotyping by liquid chromatography-mass spectrometry.

While electrospray ionization mass spectrometry has shown great potential for the identification of genotypes in DNA sequences amplified by polymerase chain reaction (PCR), the quantitative determination of allele frequencies remains challenging because of the presence of cationic adducts in the mass spectra which severely impairs accuracy of quantitation. We report here on the elaboration of an optimized desalting protocol for ion-pair reversed-phase high-performance liquid chromatography-electrospray ionization mass spectrometry (ICEMS) of PCR amplicons which facilitates the genotyping of single nucleotide polymorphisms (SNPs). Chromatographic purification at temperatures between 50 and 70 degrees C using monolithic reversed-phase columns and acetonitrile gradients in aqueous, 20-30 mmol/l butyldimethylammonium bicarbonate enabled the mass spectrometric analysis of nucleic acid solutions containing up to 1.7 mol/l sodium chloride. A further improvement in removal of metal cations was achieved upon the addition of 5-10 mmol/l ethylenediaminetetraacetic acid (EDTA) to the sample solution prior to liquid chromatography. ICEMS was used for the semi-quantitative genotyping of SNPs amplified from the tetraploid genome of potato cultivars. Using a quadrupole ion trap mass spectrometer, allele frequencies were determined with an accuracy of 2-9% by measuring the relative intensities of the signals corresponding to the molecular mass of each of the alleles in the deconvoluted mass spectra. ICEMS results correlated well with those obtained by pyrosequencing, single nucleotide primer extension, and conventional dideoxy sequencing.

Chromatography, High Pressure Liquid↗
PubMed2006

Chemically surface modified gel (CSMG): an excellent enzyme-immobilization matrix for industrial processes.

Invertase from S. cerevisiae has been immobilized on porous silica matrix, formed using sol-gel chemistry, with surface area of approximately 650 m(2)/g. The co-condensation of silica sol with 3-aminopropyl(triethoxy)silane produced an amino-chemically surface modified silica gel (N-CSMG) with a very high ligand loading of 3.6 mmol/g SiO(2); significantly higher than commercially available matrices. Surface amine groups were activated with glutaraldehyde to produce GA-N-CSMG, and invertase covalently attached by the aldehyde. Invertase was used as a model enzyme to measure the immobilizing character of the GA-N-CSMG material. Using an optimized immobilization protocol, a very high loading of 723 mg invertase per gram GA-N-CSMG is obtained; 3-200-fold higher than values published in literature. The reproducible, immobilized activity of 246,000 U/g GA-N-CSMG is also greater than any other in literature. Immobilized invertase showed almost 99% retention of free enzyme activity and no loss in catalytic efficiency. The apparent kinetic parameters K(M) and V(M) were determined using the Michealis-Menten kinetic model. K(M) of the free invertase was 1.5 times greater than that of the immobilized invertase--indicating a higher substrate affinity of the immobilized invertase. These findings show considerable promise for this material as an immobilization matrix in industrial processes.

Chemical Industry↗
PubMed2006

Trehalose as a good candidate for enriching full-length cDNAs in cDNA library construction.

It has been reported that the disaccharide trehalose is capable of increasing the thermostability and thermoactivity of reverse transcriptase, and therefore improving the length of cDNA synthesis. However, no test has been done on how the disaccharide trehalose performs in the context of the entire cDNA synthesis processes, or whether it can seamlessly integrate into the commercially available cDNA synthesis kit. In this report, we optimized a protocol to incorporate trehalose in the Stratagene's cDNA library construction kit in order to demonstrate great improvement in cDNA's length (average length of 1.8 kb in the trehalose group versus 1.0 kb in the control). Sequence analysis of the cDNA clones showed that the addition of trehalose did not increase the error rate of the RT products but greatly increase the quantity of full-length in cDNA library.

Animals↗
PubMed2004

Development of a validated liquid chromatography method for the simultaneous determination of eight fat-soluble vitamins in biological fluids after solid-phase extraction.

In the present study, a simple and rapid reversed-phase HPLC procedure has been developed for the simultaneous determination of eight fat-soluble vitamins (retinol, menadione, menaquinone, delta-tocopherol, cholecalciferol, alpha-tocopherol, alpha-tocopherol acetate and phylloquinone) in biological fluids: blood serum and urine. The analytical column, Phenomenex Luna C18 (150 mm x 4.6 mm) 3 microm, was operating at ambient temperature. Mobile phase consisted of a mixture of CH3OH-CH3CN delivered using a linear gradient, starting with a composition of 50-50% v/v and ending at 30-70% at a flow rate of 1.3 ml/min. Xanthophyll was used as internal standard (2 ng/microl). Detection and identification was performed using a photodiode array detector. Eluent monitoring was achieved at 280 nm for vitamins and 450 nm for the internal standard. However, quantitation was performed at maximum wavelength for each vitamin. Detection limits were found in the range of 1.4-6.6 ng per 20-microl injected samples, while linearity held up to 25 ng/microl. The statistical evaluation of the method was examined performing intra-day (n = 6) and inter-day calibration (n = 7) and was found to be satisfactory, with high accuracy and precision results. The biological fluids were treated using solid-phase extraction cartridges, to remove all endogenous interferences from sample matrix. The solid-phase extraction protocol was optimized in terms of retention and elution. High extraction recoveries from biological matrices: blood serum and urine, (average recovery ranging between 95 and 97.6% for blood serum and between 94.2 and 95.8% for urine) were achieved for the eight fat-soluble vitamins, using Cyclohexyl J.T. Baker SPE cartridges with methanol as eluent, requiring small volumes, 100 microl of blood serum and 100 microl of urine.

Chromatography, High Pressure Liquid↗
PubMed2006

A general method for the extraction of citrus leaf proteins and separation by 2D electrophoresis: a follow up.

With the aim of studying differentially expressed proteins as a function of abiotic and biotic stress in citrus plants, we optimized a protocol for the extraction of total leaf proteins and their 2-DE separation using commercially available immobilized pH gradient strips (IPGs) in the first dimension. Critical factors for good reproducibility of citrus leaf protein separation were identified: trichloroacetic acid (TCA)/acetone precipitation after extraction in lysis buffer, sample fractionation on narrow range overlapping IPGs and sample-cup loading at the anodic or cathodic end of the strip. The use of thiourea and a strong detergent (C7BzO) in the solubilization/rehydration buffer, coupled with the increase to 10% of SDS in the equilibration buffer before the second dimension seemed to affect positively the resolution of basic proteins. Using our protocol we resolved about 30 basic proteins on 6.3-8.3 pH range strips. Further, our protocol was successfully applied reproducibly on the analysis of control and salt exposed leaf samples of Citrus reshni Hort. Ex Tan.

Citrus↗
PubMed2005

Electroporation and transcutaneous extraction (ETE) for pharmacokinetic studies of drugs.

The therapeutic activity and toxicity of drugs often depends on the accumulation of drugs in the peripheral anatomical compartment rather than the central compartment. In the routine practice of therapeutic drug monitoring (TDM) and pharmacokinetic studies, drug concentration determined by intermittent blood sampling is used as a surrogate for calculating the drug concentration in the peripheral compartment tissues. Microdialysis, a relatively less invasive procedure, has been used for estimation of free drug levels in dermal, subcutaneous and muscle tissues. Transcutaneous extraction of drugs from the dermal tissue is a good noninvasive alternative to phlebotomy and microdialysis. This requires a technique, which can facilitate the extraction of significant and reproducible amounts of drugs from the dermal extracellular fluid (ECF) within a short sampling duration. In the present work, we assessed the feasibility of electroporation and transcutaneous extraction (ETE) method for determining the time course of drugs in dermal ECF, using salicylic acid (SA) as a test drug. Electroporation protocol was optimized based on the in vitro diffusion studies of salicylic acid across rat skin. The concentration-time profile of total SA was determined in rats after a single i.v. bolus administration. The in vivo permeability coefficient (P(in vivo)) of rat skin was determined under steady state plasma concentration of drug created by i.v. bolus followed by constant rate infusion of SA. The pharmacokinetic parameters of the drug were determined using a two-compartment pharmacokinetic model. The theoretical predicted time course of free SA in the dermal ECF after a single i.v. bolus administration was calculated using standard formulae. The concentration of free SA determined by ETE is in good agreement with that calculated using two-compartment pharmacokinetic model. This study thus provides a credible evidence for the validity of ETE technique for determining the concentration of SA in the dermal ECF.

Animals↗
PubMed2005

Sequence determinants of a transmembrane proton channel: an inverse relationship between stability and function.

The driving forces behind the folding processes of integral membrane proteins after insertion into the bilayer, is currently under debate. The M2 protein from the influenza A virus is an ideal system to study lateral association of transmembrane helices. Its proton selective channel is essential for virus functioning and a target of the drug amantadine. A 25 residue transmembrane fragment of M2, M2TM, forms a four-helix bundle in vivo and in various detergents and phospholipid bilayers. Presented here are the energetic consequences for mutations made to the helix/helix interfaces of the M2TM tetramer. Analytical ultracentrifugation has been used to determine the effect of ten single-site mutations, to either alanine or phenylalanine, on the oligomeric state and the free energy of M2TM in the absence and the presence of amantadine. It was expected that many of these mutations would perturb the M2TM stability and tetrameric integrity. Interestingly, none of the mutations destabilize tetramerization. This finding suggests that M2 sacrifices stability to preserve its functions, which require rapid and specific interchange between distinct conformations involved in gating and proton conduction. Mutations might therefore restrict the full range of conformations by stabilizing a given native or non-native conformational state. In order to assess one specific conformation of the tetramer, we measured the binding of amantadine to the resting state of the channel, and examined the overall free energy of assembly of the amantadine bound tetramer. All of the mutations destabilized amantadine binding or were isoenergetic. We also find that large to small residue changes destabilize the amantadine bound tetramer whereas mutations to side-chains of similar volume stabilize this conformation. A structural model of the amantadine bound state of M2TM was generated using a novel protocol that optimizes a structure for an ensemble of neutral and disruptive mutations. The model structure is consistent with the mutational data.

Amantadine↗
PubMed2004

Continuous measurements of cerebral tissue oxygen pressure during hyperbaric oxygenation--HBO effects on brain edema and necrosis after severe brain trauma in rabbits.

INTRODUCTION: Severe brain injury is one of the most frequent causes of severe disability in the young. In acute management of brain trauma, new approaches based on experimental animal investigations should be sought. METHODS: Twenty male, juvenile Chinchilla-Bastard rabbits received standardized cold-injury-induced-brain-trauma (CIBT). A metal probe (temperature -196 degrees C) was applied epidurally over 10 s. The hyperbaric oxygenation (HBO) group (n=10) underwent 90-min HBO sessions with 100% oxygen at 2.5 atmospheres absolute (1 h, 24+/-2 h, 48+/-2 h after CIBT). Cerebral tissue pO2-measurements were performed 60 min after CIBT, during the three HBO sessions and on day 4. The control group (n=10) underwent no treatment. Animals were sacrificed on day 4, and brains were analyzed histologically. RESULTS: In the HBO group, pO2 measurements showed a significant increase in pO2 between day 1 and day 4, whereas no significant changes were observed in the control group. During the first HBO session, mean pO2 was 169 mm Hg, during the second 305 mm Hg and during the third 420 mm Hg. The mean area of necrosis was 16.2 mm2 in the HBO group, in the control group 19.9 mm2. The areas of brain edema were significantly smaller in the HBO group. Mortality in the HBO group was 0%, in the control group 20%. CONCLUSION: HBO appears to be beneficial as an adjunct treatment of severe head trauma. To find optimal treatment protocols, further clinical studies must be developed.

Animals↗
PubMed2006

Fermentation reactor coupled with capillary electrophoresis for on-line bioprocess monitoring.

In this paper, a filter probe integrated into a computerized pneumatic sampler for capillary electroseparations was developed for an on-line monitoring of bioprocesses. The optimization of the performance of the coupled system was done by using a response surface modeling and three-level two-factor design. The resolution was found to be the most important parameter influencing the performance of an on-line analysis of the microbial metabolism. For the on-line analysis the migration time and detection limit were also found to be important parameters. Different parameters were combined by using an overall desirability function to find optimum conditions for all parameters. The equipment with an optimized separation protocol was used to monitor the bioaccumulation of Cu, Zn, Co and Cd (with detection limits 0.46, 0.37, 1.2, 0.84 mM correspondingly) by the Rhodococcus sp. bacteria isolated from the highly polluted technogenic soil of northeastern Estonia during a 2-week experiment.

Amino Acids↗