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PubMed1998

Fourier transform Raman spectroscopy of polyacrylamide gels (PAGs) for radiation dosimetry.

Polyacrylamide gels (PAGs) are used for magnetic resonance imaging radiation dosimetry. Fourier transform (FT) Raman spectroscopy studies were undertaken to investigate cross-linking changes during the copolymerization of polyacrylamide gels in the spectral range of 200-3500 cm(-1). Vibrational bands of 1285 cm(-1) and 1256 cm(-1) were assigned to acrylamide and bis-acrylamide single CH2 deltaCH2 binding modes. Bands were found to decrease in amplitude with increasing absorbed radiation dose as a result of copolymerization. Principal component regression was performed on FT-Raman spectra of PAG samples irradiated to 50 Gy. Two components were found to be sufficient to account for 98.7% of the variance in the data. Cross validation was used to establish the absorbed radiation dose of an unknown PAG sample from the FT-Raman spectra. The calculated correlation coefficient between measured and predictive samples was 0.997 with a standard error of estimate of 0.976 and a standard error of prediction of 1.140. Results demonstrate the potential of FT-Raman spectroscopy for ionizing radiation dosimetry using polyacrylamide gels.

Acrylic Resins↗
PubMed1999

Determination of acrylamide monomer in polyacrylamide degradation studies by high-performance liquid chromatography.

A high-performance liquid chromatography method using C18 and ion-exchange columns in series is developed for the determination of acrylamide and acrylic acid monomers in polymeric samples. The C18 column acts as a guard column, trapping surfactants and impurities and retaining the nonionic species. The ion-exchange column then separates the monomers according to their respective ionic strengths. This method has been proven in the laboratory to work successfully for all types of acrylamide/acrylic acid polymers and matrices. Detection limits for both monomers can be achieved in the parts-per-billion range. The method is used to study the possible degradation of polyacrylamide to acrylamide monomer in the presence of glyphosate (a herbicide) and sunlight. Polyacrylamide is used as a spray drift reduction aid in combination with glyphosate. In normal applications, the polymer and herbicide are in contact with each other in the presence of sunlight. The results show that the polymer does not degrade to acrylamide in the presence of glyphosate or sunlight or any combination of the two. It is also observed that glyphosate influences the solubility of polyacrylamide, and care must be used when combining the two.

Acrylamides↗
PubMed1978

Sequence analysis of 5'[32P] labeled mRNA and tRNA using polyacrylamide gel electrophoresis.

Sequence analysis of 5'-[32P] labeled tRNA and eukaryotic mRNA using an adaptation of a method recently described by Donis-Keller, Maxam and Gilbert for mapping guanines, adenines and pyrimidines from the 5'-end of an RNA is described. In addition, a technique utilizing two-dimensional polyacrylamide gel electrophoresis for identification of pyrimidines within a sequence is described. 5'-[32P] Labeled rabbit beta-globin mRNA and N. crassa mitochondrial initiator tRNA were partially digested with T1- RNase for cleavage at G residues, with U2-RNase for cleavage at A residues, with an extracellular RNase from B. cereus for cleavage at pyrimidine residues and with T2-RNase or with alkali for cleavage at all four residues. The 5'-[32P] labeled partial digestion products were separated according to their size, by electrophoresis in adjacent lanes of a polyacrylamide slab gel and the location of G's, A's and of pyrimidines extending 60-80 nucleotides from the 5'-end of the RNA determined. Two-dimensional polyacrylamide gel electrophoresis was used to separate the 5'-[32P] labeled fragments present in partial alkali digests of a 5'-[32P] labeled mRNA. The mobility shifts corresponding to the difference of a C residue were distinct from those corresponding to a U residue and this formed the basis of a method for distinguishing between the pyrimidines.

Animals↗
PubMed2006

Biocompatibility of two novel dermal fillers: histological evaluation of implants of a hyaluronic acid filler and a polyacrylamide filler.

BACKGROUND: Several biomaterials are currently available for soft-tissue augmentation. Biocompatibility is an indispensable condition for any such product. Appropriate histologic evaluation is a prerequisite for understanding the responses of tissues to implant materials. Recently, hyaluronic acid and polyacrylamide gel products have been introduced as dermal fillers. Both types of product are widely considered to be biocompatible. METHODS: The present study compared tissue responses in a rat in vivo model (n = 80) to a hyaluronic acid filler (Restylane Perlane; Q-Med AB, Uppsala, Sweden) and a polyacrylamide gel filler (Aquamid; Contura SA, Montreux, Switzerland). Four groups were evaluated: group 1 (n = 20) received the Restylane Perlane implant, group 2 received the Aquamid implant (n = 20), group 3 comprised a placebo group (n = 20), group 4 was the control group (n = 20). Responses and biocompatibility were assessed by histopathologic and histomorphometric evaluations between 1 week and 8 months after implantation. RESULTS: The two products induced very different tissue responses. The polyacrylamide gel filler was highly bioactive, undergoing cell infiltration and integration into tissues. The hyaluronic acid filler underwent minimal cell infiltration, and the product remained surrounded by a uniformly thin capsule. CONCLUSIONS: This study reveals that two soft-tissue fillers considered to be biocompatible induce very different tissue reactions. This indicates that their behavior in clinical practice is likely to be different.

Acrylic Resins↗
PubMed1978

Enzymatic attack on immobilized substrates. 2. Diffusional limitations in the alpha-chymotrypsin-catalyzed hydrolysis of polyacrylamide-bound l-phenylalanine 4-nitroanilide.

1. The chymotrypsin-catalyzed hydrolysis of polyacrylamide-bound L-phenylalanine 4-nitroanilide was studied. As a spacer, one or two 6-aminohexanoyl residues were inserted between the matrix and ligand. 2. In the course of the enzymatic hydrolysis of polyacrylamide-bound substrates, enzyme adsorption by the gel substrates was observed. A quasi equilibrium of enzyme partitioning was reached after approximately 20-min incubation time. The enzyme adsorption could be described by the Langmuir adsorption isotherm. 3. The substrates attached via spacers to the matrix were completely hydrolyzed. 4. The initial course of the product vs time curves, as well as the dependence of the initial hydrolysis rates on enzyme concentration or substrate concentration, have been interpreted by the Nernst reaction theory. From the results obtained it has been concluded that the inital rate of the hydrolysis of polyacrylamide-bound L-phenylalanine 4-nitroanilide depends on the velocity of enzyme diffusion into the matrix.

Acrylamides↗
PubMed2006

Directed evolution of formate dehydrogenase from Candida boidinii for improved stability during entrapment in polyacrylamide.

In two cycles of an error-prone PCR process, variants of formate dehydrogenase from Candida boidinii were created which revealed an up to 4.4-fold (440%) higher residual activity after entrapment in polyacrylamide gels than the wild-type enzyme. These were identified in an assay using single precursor molecules of polyacrylamide instead of the complete gel for selection. The stabilization resulted from an exchange of distinct lysine, glutamic acid, and cysteine residues remote from the active site, which did not affect the kinetics of the catalyzed reaction. Thermal stability increased at the exchange of lysine and glutamic acid, but decreased due the exchange of cysteine. Overall, the variants reveal very suitable properties for application in a technical synthetic process, enabling use of entrapment in polyacrylamide as an economic and versatile immobilization method.

Acrylic Resins↗
PubMed1986

Two-Dimensional Polyacrylamide Gel Analysis of Plodia interpunctella Granulosis Virus.

The structural polypeptides of purified Plodia interpunctella granulosis virus were analyzed by three different two-dimensional gel systems. Isoelectric focusing followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis allowed resolution of 53 acidic polypeptides in the enveloped nucleocapsid of the virus ranging in molecular weight from 97,300 to 8,000. Nine of these polypeptides were shown to be glycoproteins by the technique of radiolabeled lectin blotting. Separation of the granulin in this system allowed resolution of five species, all of which have identical tryptic peptide maps. This matrix protein was demonstrated to be a phosphoglycoprotein by radiolabeled lectin blotting and acid phosphatase dephosphorylation. Nonequilibrium pH gel electrophoresis followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis allowed resolution of the major basic protein of the virus, VP12, from a more acidic protein of the same molecular weight. Tryptic peptide analysis demonstrated that these two proteins were indeed different and acid urea gels followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis allowed localization of the acidic protein to the envelope and the basic protein to the nucleocapsid of the virus. Finally, probing of the separated envelope nucleocapsid proteins in both the isoelectric focusing and nonequilibrium pH gel electrophoresis two-dimensional systems after transfer to nitrocellulose with iodinated, purified viral proteins allowed further insight into reactions which may be important in the maintenance of the virion structure.

Journal Article↗
PubMed1983

Thyroid hormone increases type I adenosine 3', 5'-monophosphate-dependent protein kinase and casein kinase activities in rat liver cytosol: analysis of protein kinases by polyacrylamide disc gel electrophoresis.

We investigated the action of thyroid hormone on each protein kinase in rat liver cytosol. Kinases were analyzed by polyacrylamide disc gel electrophoresis and isoelectric focusing in polyacrylamide gel. Polyacrylamide disc gel electrophoresis separated cAMP-dependent protein kinase type I (Rf = 0.35), type II (Rf = 0.44), their catalytic subunit (Rf = 0.26), and cAMP-independent protein kinase (Rf = 0.50). Casein kinase was detected at Rf = 0.37. In addition to the catalytic subunit with Rf = 0.26, another catalytic subunit was found at Rf = 0.44 when the cytosol was preincubated with cAMP. The administration of T3 (20 micrograms/100 g BW for 3 days) to hypothyroid rats increased enzyme activities of type I holoenzyme and casein kinase by 48%. Free catalytic subunit, separated from holoenzyme, had the same level of enzyme activity in both groups, suggesting greater endogenous dissociation of type I holoenzyme in hypothyroid rats. When heat-inactivated rat liver cytosol was used as substrate in the assay of protein kinase activity, the peak enzyme active in phosphorylating the cytosol corresponded to the casein kinase peak. Our data indicate that casein kinase is the main enzyme that mediates phosphorylation of endogenous proteins in rat liver cytosol, and that T3 treatment increases the activity of casein kinase and of type I cAMP-dependent protein kinase.

Animals↗
PubMed2005

Bulk and micropatterned conjugation of extracellular matrix proteins to characterized polyacrylamide substrates for cell mechanotransduction assays.

Increasing numbers of cell mechanotransduction studies are currently utilizing elastic substrates fabricated from polyacrylamide in the form of thin gels. Their versatility depends on the ability to ensure the appropriate gel stiffness and control the uniformity and geometry of extracellular matrix protein coating of the gel. Beginning with a brief quantitative emphasis on the elastic properties of polyacrylamide gels, we present an inexpensive and highly reproducible method for uniform coating with a wide variety of extracellular matrix proteins. We used a reducing agent, hydrazine hydrate, to modify nonreactive amide groups in polyacrylamide to highly reactive hydrazide groups that can form covalent bonds with aldehyde or ketone groups in oxidized proteins. This simple conjugation method overcomes the limitations of previously used photoactivatable cross-linkers: nonuniform coating due to nonuniformity of irradiation and technically challenging procedures for micropatterning. As demonstrated in our study of cell polarity during constrained migration, this conjugation method is especially effective in gel micropatterning by manual microcontact printing of protein patterns as small as 5 microm and enables numerous studies of constrained cell attachment and migration that were previously unfeasible due to high cost or difficulty in controlling the protein coating.

Acrylic Resins↗
PubMed2004

[Applications of magnetic resonance imaging in diagnosis of the complications after breast augmentation with polyacrylamide hydrogel injection].

OBJECTIVE: To explore the clinical value of magnetic resonance (MR) imaging in the diagnosis of the complications after breast augmentation with polyacrylamide hydrogel injection. METHODS: MRI was performed in 16 patients who had breast augmentation with polyacrylamide hydrogel injection. The results of magnetic resonance imaging were compared with the clinical symptoms. RESULTS: MRI examinations of the 16 patients showed that (1) 4 breasts in 2 patients were comparatively natural; (2) a large lacteal cyst was detected in one patients in lactation; (3) breast asymmetry occurred in 3 patients (6 sides); (4) in 10 patients (20 sides), the injected gel was in irregular form. The results of MRI were in accord with the clinical symptoms. CONCLUSION: MRI is of great diagnostic value for the complications after breast augmentation with polyacrylamide hydrogel injection.

Acrylic Resins↗
PubMed1995

A study of collagen-hyaluronan interaction through swelling in polyacrylamide gels.

The equilibrium swelling of biopolymer loaded polyacrylamide gels can yield information on polymer-polymer interactions. Using the temperature dependence of excess equilibrium swelling, equations were derived to yield homopolymer as well as heteropolymer interactions. Hyaluronan and collagen (Type I) encapsulated in polyacrylamide gels showed increasing isothermal swelling with increase in the bipolymer concentration. The pH dependence of isothermal swelling caused by hyaluronan indicated that the swelling was largely due to repulsion of the charges of the carboxylate groups. Temperature dependent studies showed a decrease in swelling with an increase in temperature for collagen. The opposite trend was found for hyaluronan. This indicates that the interactions among collagen monomers are largely hydrophobic, while the interactions among hyaluronan molecules are mainly hydrogen bonding and repulsive ionic forces. Combined hyaluronan and collagen in polyacrylamide gels demonstrated an increase in swelling with an increase in temperature albeit much reduced compared with hyaluronan alone. Collagen interacts with hyaluronan through ionic attraction.

Acrylic Resins↗
PubMed1996

Activation energy of the separation of DNA sequencing fragments in denaturing noncross-linked polyacrylamide by capillary electrophoresis.

We present the mobility of DNA sequencing fragments as a function of temperature in 5%T, 0%C noncross-linked polyacrylamide and at an electric field of 150 V/cm. The mobility of the fragments follows the Arrhenius equation. The activation energy for migration of a fragment through the polymer decreases monotonically with fragment length. The data were also analyzed in terms of the biased reptation model; the onset of biased reptation with stretching occurs for longer fragments as the temperature increase. These results are quite different from those observed for separation of DNA in cross-linked gels and represent a fundamental difference in the physics of the cross-linked and noncross-linked polyacrylamide.

Acrylic Resins↗
PubMed2003

Size-based separations of proteins by capillary electrophoresis using linear polyacrylamide as a sieving medium: model studies and analysis of cider proteins.

Electrophoretic conditions to separate sodium dodecyl sulfate (SDS)-protein complexes according to their relative molecular mass by capillary electrophoresis (CE) using linear polyacrylamide as a sieving matrix were examined. Five purified proteins with relative molecular masses between 14 400 and 66 200 Da were separated on a coated fused-silica capillary with an internal diameter of 100 microm and an effective length of 24 cm (total length, 32.5 cm). Benzoic acid was added to the solution of purified proteins as internal standard; beta-mercaptoethanol was also added as reducing agent. The running buffer composition was 0.05 M tris(hydroxymethyl)aminomethane (Tris), 0.035 M aspartic acid, 0.1% m/v SDS, 4% m/v acrylamide, the resulting pH being 8.0. The applied voltage was 7 kV (reversed voltage polarity) in order to avoid high current intensities. Under optimized conditions, the five proteins were separated in less than 15 min, with a % relative standard deviation (RSD) between 0.2 and 0.4 for migration times in the same day. Good efficiency (values between 150 000 and 40 000 N/m) and resolution (values between 2 and 2.8) were obtained. The inverse of relative migration times was found to correlate with the logarithm of their relative molecular mass. Finally, cider proteins were analyzed and their relative molecular masses were determined. These results were compared with those obtained by SDS-polyacrylamide gel electrophoresis (SDS-PAGE).

Acrylic Resins↗
PubMed1998

Detection of lectins using ligand blotting and polyacrylamide-type glycoconjugate probes.

A sensitive and convenient method for detection of the carbohydrate-binding activity of lectins was established using the combination of blotting of lectins on polyvinylidene difluoride membranes, carbohydrate-conjugated biotinylated polyacrylamide-type probes (carbohydrate-bp probes), horseradish peroxidase-streptavidin, and detection by enhanced chemiluminescence of the enzyme reaction. This method was tested for detection of four plant lectins blotted on the membrane: concanavalin A was detectable down to 100 ng by mannose-bp probe, Ricinus communis agglutinin 120 to as low as 5 ng by N-acetyllactosamine-bp probe, soybean agglutinin to 1 microgram by beta-N-acetyl-D-galactosamine-bp probe, and wheat germ agglutinin to 5 ng by beta-N-acetyl-D-glucosamine-bp probe. All four lectins were detectable on an electroblotted membrane after SDS-polyacrylamide gel electrophoresis. This method was used to detect recombinant human galectin-3 in Escherichia coli cell lysates and mannan-binding protein in human serum. These results indicate that this method is widely applicable to convenient detection and characterization of lectins in crude samples.

Acrylic Resins↗
PubMed1992

Effect of total percent polyacrylamide in capillary gel electrophoresis for DNA sequencing of short fragments. A phenomenological model.

Polyacrylamide capillary gels were prepared with constant (5% C) cross-linker concentration and with total acrylamide concentration ranging from 2.5 to 6% T. At each acrylamide concentration, peak spacing was constant for DNA sequencing fragments ranging from 25 to 250 nucleotides in length. Peak spacing increased linearly with the total acrylamide concentration. The intercept of the retention time vs. fragment length plot was independent of % T. Ferguson plots were constructed for short DNA fragments; the polyacrylamide pore size falls in the 2.5 to 3.5 nm range for the gels studied. Theoretical plate count is independent of total acrylamide concentration; longitudinal diffusion, and not thermal gradients, limit the plate count. A phenomenological model is presented that predicts retention time, plate count, and resolution for sequencing fragments ranging in size from 25 to 250 bases and gels that range from 2.5 to 6% total acrylamide.

Acrylic Resins↗
PubMed1993

DNA sequencing by capillary electrophoresis with replaceable linear polyacrylamide and laser-induced fluorescence detection.

Replaceable linear polyacrylamide (LPA) has been utilized as a sieving matrix for DNA sequencing by capillary electrophoresis (CE). Difficulties associated with cross-linked polyacrylamide gel stability have been overcome for the routine application of CE to DNA sequencing. A simple laser-induced fluorescence (LIF) detection system based on a single laser and two photomultipliers (PMT) has been adopted for this work. Sequencing information for four bases has been obtained from two fluorescent dyes and two peak height ratios, detected in two optical channels. FAM- and JOE-labeled M13 (-21) primers have been chosen because both dyes are efficiently excited with a low-power argon ion laser, can be optically separated, and exhibit minimal dye-based shifts in DNA fragment mobilities. Addition of denaturants to the electrophoresis running buffer (1 x TBE, 3.5 M urea, 30% formamide) and column operation at 32 degrees C permitted the resolution of difficult compressed sites in the sequence of phage M13mp18. Careful examination of the polymerization reaction of LPA has led to methodology that has proven to be reproducible for obtaining DNA sequencing information of M13mp18 phage for 350 nucleotides in close to 30 min.

Acrylic Resins↗
PubMed2001

Fast separation of DNA sequencing fragments in highly alkaline solutions of linear polyacrylamide using electrophoresis in bare silica capillaries.

An optimized procedure for the fast separation of DNA sequencing fragments in short bare fused-silica capillaries filled with highly alkaline solutions of replaceable linear polyacrylamide is presented. High denaturing abilities of the separation media at pH values over 12 are the main reason for their applications in analyses of ssDNA fragments. Moreover, the alkaline solutions of polyacrylamide provide other advantageous properties: three times higher electrophoretic mobility of ssDNA fragments in comparison to those in urea, negligibly low electroosmotic flow in uncoated capillaries, and an adequate stability to a fast alkaline hydrolysis. The separation power of this procedure is enhanced strongly by using monocarboxy poly(ethylene glycol), a terminator for transient isotachophoresis, which eliminates the electromigration dispersion. A high separation efficiency of our system enables to reduce analysis time to several minutes by decreasing the effective lengths of capillaries to 7 cm. A special sample introduction by diffusion is successfully applied. The experimental results demonstrate a potential of the alkaline electrolytes for an implementation in diagnostic sequencing practice.

Acrylic Resins↗
PubMed2002

Activity staining of glutathione peroxidase after electrophoresis on native and sodium dodecyl sulfate polyacrylamide gels.

Glutathione peroxidase (GSH-Px), from commercial bovine erythrocytes or ammonium sulfate fractionations (30-45%, 45-60%, 60-75% and 75-90% saturations) of ginger rhizome, was detected on polyacrylamide gels after native polyacrylamide gel electrophoresis (PAGE) or sodium dodecyl sulfate (SDS)-PAGE. The gel was submerged in a 50 mM Tris-HCl buffer (pH 7.9) containing 13 mM glutathione and 0.004% hydrogen peroxide with gentle shaking for 10-20 min. The GSH-Px activity was stained with a solution containing 1.2 mM 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 1.6 mM phenazine methosulfate (PMS) for 10 min. The clear zone of GSH-Px activity on a purple background was found in both native and SDS-PAGE gels. This fast and sensitive method can be used in the process of enzyme purification and characterization of mammalian or plant cells.

Animals↗