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PubMed1999

Synthesis of superporous hydrogels: hydrogels with fast swelling and superabsorbent properties.

We have been interested in the synthesis of hydrogels with fast swelling kinetics and superabsorbent properties. To increase the water absorption rate, interconnected pores were introduced to the hydrogels. Since the pore size in the dried hydrogels is in the order of hundreds of micrometers, these hydrogels are called "superporous" hydrogels. Superporous hydrogels were synthesized by crosslinking polymerization of various vinyl monomers in the presence of gas bubbles formed by the chemical reaction of acid and NaHCO3. The polymerization process was optimized to capture the gas bubbles inside the synthesized hydrogels. The use of the NaHCO3/acid system allowed easy control of timing for gelation and foam formation. We found that PF127 was the best foam stabilizer for most of the monomer systems used in our study. Scanning electron microscope (SEM) pictures showed interconnected pores forming capillary channels. The capillary channels, which were critical for fast swelling, were preserved during drying by dehydrating water-swollen hydrogels with ethanol before drying. The ethanol-dehydrated superporous hydrogels reached equilibrium swelling within minutes. The equilibrium swelling time could be reduced to less than a minute with the use of a wetting agent. In our study, water moisture was used as a wetting agent since the amount of moisture content in the dried hydrogels easily could be controlled. Preparation of superporous hydrogels using the right blowing system, foam stabilizer, drying method, and wetting agent makes it possible to reduce the swelling time to less than a minute regardless of the size of the dried gels. The superporous hydrogels can be used where fast swelling and superabsorbent properties are critical.

Absorption↗

PubMed1998

Elucidation of the genetic basis of the antigen presentation defects in the mutant cell line .220 reveals polymorphism and alternative splicing of the tapasin gene.

Antigenic peptides are presented to cytotoxic T lymphocytes by heterodimers of MHC class I molecules and beta2-microglobulin. Peptides are generated in the cytosol and translocated into the endoplasmic reticulum (ER) through the transporter associated with antigen processing (TAP). Optimal binding of peptides to class I molecules is facilitated by the physical association between class I heterodimers and TAP. This association is mediated largely by the glycoprotein tapasin. Analysis of tapasin function has relied on a mutant cell line, .220, which is defective in tapasin expression and antigen presentation. We have investigated the genetic basis of these defects. In .220 cells, Tapasin transcripts lack exon two. This is caused by a single nucleotide substitution, disrupting the 5' splice site of the second intron. A tapasin protein is produced in .220 cells, but has a truncated signal peptide and lacks the N-terminal 49 amino acids encoded by full-length transcripts. Nonetheless, this truncated form is translocated into the ER and interacts with TAP. As a result of alternative splicing, transcripts lacking exon two are also present in wild-type cells, although no truncated protein was detected. Additionally we describe a polymorphism in the Tapasin gene, with two alleles encoding arginine or threonine at peptide position 240.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2000

Optimization of repeated-batch transcription for RNA production.

A major obstacle to large-scale RNA production is the high raw material cost. This work focuses on reducing the cost of RNA produced by in vitro transcription. RNA can be produced by transcription from DNA templates immobilized on solid supports such as agarose beads, with yields comparable to traditional solution-phase transcription. The advantage of immobilized DNA is that the templates can be recovered from the reaction and reused in multiple rounds, eliminating unnecessary disposal. Additionally, approximately 50% of the original RNA polymerase added to the reaction is also recovered in active form with the DNA and can be used for further rounds of repeated-batch transcription. Thus, adding only a fraction of the first-round enzyme concentration to subsequent rounds is sufficient for maintaining yields comparable to batch reactions for many rounds, with lowered cost. Results for two different DNA templates support a simplified model for repeated-batch transcription, based on the previous work of Davis and Breckenridge (J Biotechnol 1999;71:25-37). The model successfully predicts the yields for several of rounds of repeated-batch transcription using various enzyme addition schemes, and it was used to optimize the process by reducing the cost of raw materials per amount of RNA produced by 40-70%.

Base Sequence↗

PubMedDate not supplied

Mandible reconstruction with microvascular surgery.

Microvascular surgery has become the preferred method for mandible reconstruction. Whenever possible, immediate reconstruction at the time of segmental mandible resection will provide the best aesthetic and functional result. Four donor sites (fibula, iliac crest, radial forearm, and scapula) have become the primary sources of vascularized bone and soft tissue for the reconstruction. The fibula has multiple advantages, including bone length and thickness, donor site location permitting flap harvest simultaneously with tumor resection, and minimal donor site morbidity. The fibula donor site should be the first choice for most defects, particularly those with anterior or large bony defects requiring multiple osteotomies. Use of an alternative donor site is best reserved for cases with large soft tissue and minimal bone requirements. Dental rehabilitation through the use of prostheses and osseointegrated dental implants is an important part of the reconstructive process to optimize aesthetics and function. An algorithm for mandible reconstruction with microvascular osseous flaps is presented. Semin. Surg. Oncol. 19:226-234, 2000.

Bone Transplantation↗

PubMed2002

Multiwell in-gel protein digestion and microscale sample preparation for protein identification by mass spectrometry.

In-gel peptide digestion has become a widely used technique for characterizing proteins resolved by two-dimensional gel electrophoresis. Peptides generated from gel pieces are frequently contaminated with detergent and salts. Prior to matrix-assisted laser desorption/ionization-time of flight mass spectrometry analysis, these contaminants are removed using micro scale C18 sample preparation columns. In this paper, data are presented to demonstrate the application of a solvent resistant MultiScreen 96-well plate with a low peptide binding membrane and ZipTip micropipette based sample preparation. Recoveries of peptides (m/z of 1000 to 5000 Da) derived from standard protein protease digests, were estimated at various stages of the analytical process. An optimized protocol has been established and all the reagents and consumables have been packaged in a ready to use commercial kit. Data will be presented to show the application of this technology package to accelerate the throughput of protein characterization by protease fragmentation.

Blood Protein Electrophoresis↗

PubMed2005

The influence of weight with assay error on gentamicin pharmacokinetics using the Bayesian and nonlinear least square regression analysis in appendicitis patients.

The purpose of this study was to determine the influence of weight with gentamicin assay error on the Bayesian and nonlinear least squares regression analysis in 12 Korean appendicitis patients. Gentamicin was administered intravenously over 0.5 h every 8 h. Three specimens were collected 48 h after the first dose from all patients at the following times, just before the regularly scheduled infusion, at 0.5 h and 2 h after the end of the 0.5 h infusion. Serum gentamicin levels were analysed by fluorescence polarization immunoassay technique with TDxFLx. The standard deviation (SD) of the assay over its working range had been determined at the serum gentamicin concentrations of 0, 2, 4, 8, 12 and 16 microg/ml in quadruplicate. The polynominal equation of gentamicin assay error was found to be SD (microg/ml) = 0.0246-(0.0495C) + (0.00203C(2)). There were differences in the influence of weight with gentamicin assay error on pharmacokinetic parameters of gentamicin using the nonlinear least squares regression analysis but there were no differences on the Bayesian analysis. This polynominal equation can be used to improve the precision of fitting of pharmacokinetic models to optimize the process of model simulation both for population and for individualized pharmacokinetic models. The result would be improved dosage regimens and the better, safer care of patients receiving gentamicin.

Anti-Bacterial Agents↗

PubMed2004

Transient production of recombinant proteins by Chinese hamster ovary cells using polyethyleneimine/DNA complexes in combination with microtubule disrupting anti-mitotic agents.

We have developed a simple and robust transient expression system utilizing the 25 kDa branched cationic polymer polyethylenimine (PEI) as a vehicle to deliver plasmid DNA into suspension-adapted Chinese hamster ovary cells synchronized in G2/M phase of the cell cycle by anti-mitotic microtubule disrupting agents. The PEI-mediated transfection process was optimized with respect to PEI nitrogen to DNA phosphate molar ratio and the plasmid DNA mass to cell ratio using a reporter construct encoding firefly luciferase. Optimal production of luciferase was observed at a PEI N to DNA P ratio of 10:1 and 5 mug DNA 10(6) cells(-1). To manipulate transgene expression at mitosis, we arrested cells in G2/M phase of the cell cycle using the microtubule depolymerizing agent nocodazole. Using secreted human alkaline phosphatase (SEAP) and enhanced green fluorescent protein (eGFP) as reporters we showed that continued inclusion of nocodazole in cell culture medium significantly increased both transfection efficiency and reporter protein production. In the presence of nocodazole, greater than 90% of cells were eGFP positive 24 h post-transfection and qSEAP was increased almost fivefold, doubling total SEAP production. Under optimal conditions for PEI-mediated transfection, transient production of a recombinant chimeric IgG4 encoded on a single vector was enhanced twofold by nocodazole, a final yield of approximately 5 microg mL(-1) achieved at an initial viable cell density of 1 x 10(6) cells mL(-1). The glycosylation of the recombinant antibody at Asn297 was not significantly affected by nocodazole during transient production by this method.

Animals↗

PubMed2005

Removal of poly-histidine fusion tags from recombinant proteins purified by expanded bed adsorption.

Enzymatic methods have been used to cleave the C- or N-terminus polyhistidine tags from histidine tagged proteins following expanded bed purification using immobilized metal affinity chromatography (IMAC). This study assesses the use of Factor Xa and a genetically engineered exopeptidase dipeptidyl aminopeptidase-1 (DAPase-1) for the removal of C-terminus and N-terminus polyhistidine tags, respectively. Model proteins consisting of maltose binding protein (MBP) having a C- or N-terminal polyhistidine tag were used. Digestion of the hexahistidine tag of MBP-His(6) by Factor Xa and HT15-MBP by DAPase-1 was successful. The time taken to complete the conversion of MBP-His(6) to MBP was 16 h, as judged by SDS-PAGE and Western blots against anti-His antibody. When the detagged protein was purified using subtractive IMAC, the yield was moderate at 71% although the overall recovery was high at 95%. Likewise, a yield of 79% and a recovery of 97% was obtained when digestion was performed with using "on-column" tag digestion. On-column tag digestion involves cleavage of histidine tag from polyhistidine tagged proteins that are still bound to the IMAC column. Digestion of an N-terminal polyhistidine tag from HT15-MBP (1 mg/mL) by the DAPase-I system was superior to the results obtained with Factor Xa with a higher yield and recovery of 99% and 95%, respectively. The digestion by DAPase-I system was faster and was complete at 5 h as opposed to 16 h for Factor Xa. The detagged MBP proteins were isolated from the digestion mixtures using a simple subtractive IMAC column procedure with the detagged protein appearing in the flowthrough and washing fractions while residual dipeptides and DAPase-I (which was engineered to exhibit a poly-His tail) were adsorbed to the column. FPLC analysis using a MonoS cation exchanger was performed to understand and monitor the progress and time course of DAPase-I digestion of HT15-MBP to MBP. Optimization of process variables such as temperature, protein concentration, and enzyme activity was developed for the DAPase-I digesting system on HT15-MBP to MBP. In short, this study proved that the use of either Factor Xa or DAPase-I for the digestion of polyhistidine tags is simple and efficient and can be carried out under mild reaction conditions.

Carrier Proteins↗

PubMed2007

Alginate microparticles as novel carrier for oral insulin delivery.

Alginate microparticles produced by emulsification/internal gelation were investigated as a promising carrier for insulin delivery. The procedure involves the dispersion of alginate solution containing insulin protein, into a water immiscible phase. Gelation is triggered in situ by instantaneous release of ionic calcium from carbonate complex via gentle pH adjustment. Particle size is controlled through the emulsification parameters, yielding insulin-loaded microparticles. Particle recovery was compared using several washing protocols. Recovery strategies are proposed and the influence on particle mean size, morphology, recovery yield (RY), encapsulation efficiency, insulin release profile, and structural integrity of released insulin were evaluated. Spherical micron-sized particles loaded with insulin were produced. The recovery process was optimized, improving yield, and ensuring removal of residual oil from the particle surface. The optimum recovery strategy consisted in successive washing with a mixture of acetone/hexane/isopropanol coupled with centrifugation. This strategy led to small spherical particles with an encapsulation efficiency of 80% and a RY around 70%. In vitro release studies showed that alginate itself was not able to suppress insulin release in acidic media; however, this strategy preserves the secondary structure of insulin. Particles had a mean size lower than the critical diameter necessary to be orally absorbed through the intestinal mucosa followed by their passage to systemic circulation and thus can be considered as a promising technology for insulin delivery.

Administration, Oral↗

PubMed2004

Evidence for the disproportionate mapping of olfactory airspace onto the main olfactory bulb of the hamster.

Olfactory receptor neurons (ORNs) project to the rodent main olfactory bulb (MOB) from spatially distinct air channels in the olfactory recesses of the nose. The relatively smooth central channels of the dorsal meatus map onto the dorsal MOB, whereas the highly convoluted peripheral channels of the ethmoid turbinates project to the ventral MOB. Medial and lateral components of each projection stream innervate the medial and lateral MOB, respectively. To ascertain whether such topography entails the disproportionate representation seen in other sensory maps, we used disector-based stereological techniques in hamsters to estimate the number of ORNs associated with each channel in the nose and the number of their targets (glomeruli and mitral and tufted cells) in corresponding divisions of the MOB. Each circumferential half of the MOB (dorsal/ventral, medial/lateral) contained about 50% of the 3,100 glomeruli and about 50% of the 160,000 mitral and tufted cells per bulb. We found equivalent numbers of ORNs with dendritic knobs in the medial and lateral channels (4.5 million each). However, the central channels had only 2 million knobbed ORNs, whereas the peripheral channels had 7 million. Thus, there is a disproportionate mapping of the central-peripheral axis of olfactory airspace onto the dorsal-ventral axis of the MOB, encompassing a greater than threefold variation in the average convergence of ORNs onto MOB secondary neurons. We hypothesize that the disproportionate projections help to optimize chemospecific processing by compensating, with differing sensitivity, for significant variation in the distribution and concentration of odorant molecules along the olfactory air channels during sniffing.

Animals↗

PubMed2003

Separation of aminoalkanephosphonic acid enantiomers by indirect UV detection capillary electrophoresis with application of cyclodextrins.

Indirect UV detection capillary electrophoresis (CE) was used for the separation of aminoalkanephosphonic acid (AP) enantiomers by applying commercially available cyclodextrins as chiral discriminators. The results show that the separation of the enantiomers depends on pH of the background electrolyte, the molar ratio of cyclodextrin to aminophosphonic acid, and on the type of the applied chiral selector. Optimization of process conditions allowed enantiomeric baseline separation or partial separation of 12 out of 14 alpha-aminophosphonic acids studied. This type of CE might therefore be successfully used for routine determination of enantiomeric purity of aminophosphonic acids.

Amines↗

PubMed2006

Enantiomeric screening of racemic citalopram and metabolites in human urine by entangled polymer solution capillary electrophoresis: an innovatory robustness/ruggedness study.

Several CE methods have been developed to achieve the chiral separation of citalopram (CIT) and its metabolites demethylcitalopram (DCIT), didemethylcitalopram (DDCIT), and citalopram N-oxide (CIT-NO). All of these compounds were present as racemic mixtures. The best method, which led to the first ever chiral screening of CIT, DCIT, DDCIT, and CIT-NO, involved the use of carboxymethyl-gamma-CD (CM-gamma-CD) and the entangled polymer hydroxypropylmethylcellulose (HPMC) as chiral and selectivity additives, respectively, in the buffer system. In an effort to improve the selectivity and sensitivity of the method, the chemical and instrumental parameters were optimized. The best conditions were short-end anodic hydrodynamic injection (6 s, 0.7 psi); as BGE pH 5, 20 mM phosphate buffer, 0.2% w/v CM-gamma-CD, 0.05% w/v HPMC; voltage of 28 kV with a ramp applied (0.4 s); cartridge temperature of 20 degrees C; detection at 205 nm. In addition, a simple and rapid achiral CE method for the determination of citalopram propionic acid (CIT-PA, the only anionic metabolite of CIT) is also reported for the first time. Prior to the electrophoretic procedure it was necessary to apply an extraction and preconcentration step to obtain analytes from the human urine samples. This was achieved using an optimized SPE process. Moreover, an innovatory experimental and statistical design approach, which involves the simultaneous evaluation of the global robustness and ruggedness effects, was applied. Both of the proposed methods proved to be very useful in the chiral pharmacokinetic screening of CIT and related metabolites in clinical human urine samples.

Antidepressive Agents, Second-Generation↗

PubMed2006

Development of a capillary electrophoresis-mass spectrometry method for the determination of formaldehyde releasers as their hydrolysis products and amino alcohols from metal working fluids.

Metal working fluids (MWFs) are widely used as lubricants and coolants for different industrial operations. Biocides are ingredients of MWFs to control the microbial growth; derivatives of hexahydrotriazines and oxazolidines are generally used. Because of the lack of appropriate characterization, an existing capillary electrophoretic method for their quantification was improved. During the process of optimization, it became clear that hydrolysis products, derivatives of amino alcohols, severely interfere with the separation procedure. Since indirect-UV detection lacked the required selectivity, mass-selective detection was employed. NMR and MS established the absence of amino alcohols in the original educts. The aqueous solutions of the biocides stored for extended time remained amino alcohol-free, suggesting that these amino alcohols are formed from the biocides during the capillary electrophoretic separation. The observation of narrow and symmetric peaks indicated hydrolysis, and the polarity of the products implied favorable conditions for capillary electrophoretic separation. Methods were optimized for the analysis of the amino alcohols, the hydrolytic products of the formaldehyde releasers, using indirect-UV and MS detection. This method was extended to other likely solutes used as alkaline-reserve ingredients. The analytes were separated within 9 min with a high precision of migration times (the RSDs were below 1.5%). When quantifying from mobility scale, the calibration curves produced linearity with regression coefficients in the range of 0.990-0.999. The detection limit was lower than 1 mg/L in the case of MS detection. The influence of water-based MWF was also investigated, and no matrix effect on the migration of the analytes and on the peak areas was observed.

Amino Alcohols↗

PubMed2006

Motion correction and the use of motion covariates in multiple-subject fMRI analysis.

The impact of using motion estimates as covariates of no interest was examined in general linear modeling (GLM) of both block design and rapid event-related functional magnetic resonance imaging (fMRI) data. The purpose of motion correction is to identify and eliminate artifacts caused by task-correlated motion while maximizing sensitivity to true activations. To optimize this process, a combination of motion correction approaches was applied to data from 33 subjects performing both a block-design and an event-related fMRI experiment, including analysis: (1) without motion correction; (2) with motion correction alone; (3) with motion-corrected data and motion covariates included in the GLM; and (4) with non-motion-corrected data and motion covariates included in the GLM. Inclusion of covariates was found to be generally useful for increasing the sensitivity of GLM results in the analysis of event-related data. When motion parameters were included in the GLM for event-related data, it made little difference if motion correction was actually applied to the data. For the block design, inclusion of motion covariates had a deleterious impact on GLM sensitivity when even moderate correlation existed between motion and the experimental design. Based on these results, we present a general strategy for block designs, event-related designs, and hybrid designs to identify and eliminate probable motion artifacts while maximizing sensitivity to true activations.

Adolescent↗

PubMed2005

Fabrication and evaluation of nanoporous alumina membranes for osteoblast culture.

An understanding of osteoblast response to surface topography is essential for successful bone tissue engineering applications. Alumina has been extensively used as a substrate for bone tissue constructs. However, current techniques do not allow precise surface topography and orientation of the material. In this research, a two-step anodization process was optimized for the fabrication of nanoporous alumina membranes with uniform pore dimension and distribution. The anodization voltage can be varied to create nanoporous alumina membranes with pore sizes ranging from 30 to 80 nm in diameter. The impact of the nanoscale pores on osteoblast response was studied by evaluating cell adhesion, morphology, and matrix production. Scanning electron microscopy and atomic force microscopy were used to characterize the nanoporous alumina membranes. Osteoblast adhesion and morphology were investigated using scanning electron microscopy images and matrix production was characterized using energy dispersive spectroscopy. This research combined the advantages of using alumina, a material with proven biocompatibility and current orthopedic clinical applications, and incorporated porous features on the nanoscale which have been reported to improve osteoblast response.

Aluminum Oxide↗

PubMed2006

Adsorption of bisphosphonate onto hydroxyapatite using a novel co-precipitation technique for bone growth enhancement.

Premature bone resorption and remodeling by osteoclasts can limit the longevity of implant fixation and recovery time. Orally administered bisphosphonates (BPs) have been used to inhibit osteoclast action at the implant/bone interface. Ideally, these should be delivered at the interface with the osteoblast-active hydroxyapatite (HA) for maximum effect. This investigation introduces a novel BP loading technique to achieve improved BP release from a simulated body fluid-grown HA (SBF-HA) with the aim of improving implant fixation. A solution co-precipitation technique incorporates the BP (pamidronate) into a thin SBF-HA coating. Surface analysis, using X-ray photoelectron spectroscopy (XPS), of the resultant coating was employed to confirm the presence of the adsorbed BP on the surface of SBF-HA. XPS analysis was also used to determine the optimal adsorption process. Osteoclast cell culture experiments confirmed the biological effectiveness of BP adsorption and proved that the pamidronate was biologically active, causing both decreased osteoclast numbers and decreased resorption.

Adsorption↗

PubMed1993

Covalent immobilization of aminoacylase to alginate for L-phenylalanine production.

Aminoacylase I (EC. 3.5.1.14) was immobilized by covalent crosslinking to alginate molecules with 1-ethyl-3-(3-dimethyl-aminopropyl)-carbodiimide HCl followed by calcium alginate bead formation for the production of L-phenylalanine from the racemic mixtures of N-acetyl-DL-phenylalanine. Different concentrations of the coupling reagent were tested and the coupling process was optimized. The immobilized and the partially purified aminoacylase were characterized in terms of the activity, operational stability, thermal stability, pH and temperature optima and kinetic constants, Km and Vmax. The activity of the enzyme covalently immobilized in calcium alginate beads was enhanced by about 75% compared to that of free enzyme. The beads showed stable activity under operational conditions, they lost about 40% of their activity after four reaction cycles. The immobilized aminoacylase was more stable over a broader pH range. Thus this simple method provides irreversible immobilization of aminoacylase to give a biocatalyst with good operational stability and enhanced activity.

Alginates↗

PubMed1986

Formation and characterization of cisplatin loaded poly(d,l-lactide) microspheres for chemoembolization.

Cisplatin, a slightly water soluble anticancer drug, has been incorporated into biodegradable poly(d,l-lactide) microspheres using the solvent evaporation process. The optimal experimental conditions to produce spherical and separate drug-loaded particles (45% cisplatin) were as follows: the dispersing phase was a mixture of 0.05% methylcellulose and 4% polyvinyl alcohol (8 mPa-s grade); and the optimal poly(d,l-lactide) concentration in the organic phase was found to be greater than or equal to 7.16%. Microscopic studies showed that increasing the drug content in the microspheres produced the appearance of rod-like crystals at the microparticle surface. In addition, the cisplatin crystals were found homogeneously distributed in the polymer matrix, even at a high drug content. Increased viscosities of the organic phase enhanced the mean microsphere size, while increasing the emulsifier concentration in the aqueous phase decreased the average particle size. The drug incorporation efficiency was markedly improved after saturation of the dispersing phase with cisplatin. It was also noted that the amount of drug incorporated increased with increasing mean microsphere diameter. The methylene chloride content entrapped within the microspheres was found to depend upon the microsphere size distribution and the cisplatin content. An increase of the microsphere system porosity, by the addition of 10% cyclohexane in the organic phase, caused a reduction in the residual methylene chloride content. Finally, the in vitro release kinetics of cisplatin were influenced by the drug loading.

Chemistry, Pharmaceutical↗