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PubMed1989

Bactericidal concentrations of chlorhexidine-digluconate, amine fluoride gel and stannous fluoride gel for subgingival bacteria tested in serum at short contact times.

In vitro inhibitory and bactericidal concentrations in serum of chlorhexidine-digluconate, amine fluoride gel, stannous fluoride gel, stannous fluoride, metronidazole and amoxicillin were determined against Bacteroides gingivalis, Bacteroides intermedius, Fusobacterium nucleatum, Actinobacillus actinomycetemcomitans and Capnocytophaga sputigena. The minimal inhibitory concentration was assessed by the agar dilution technique. The killing curves and minimal bactericidal concentration of the antimicrobial agents in inactivated bovine serum were determined after 5, 10, 20 and 60 minutes contact time. The minimal inhibitory concentration varied amongst the tested bacteria. A concentration of 128 micrograms/ml chlorhexidine digluconate, 20 mg/ml amine fluoride gel, 1 mg/ml stannous fluoride, 128 micrograms/ml metronidazole and 4 micrograms/ml amoxicillin inhibited the growth of the tested species. The minimal bactericidal concentration in serum for B. gingivalis, B. intermedius, F. nucleatum, A. actinomycetemcomitans and C. sputigena after 10 min contact time was 5 mg/ml for chlorhexidine digluconate and 100 mg/ml for amine fluoride gel. A concentration of 200 mg/ml stannous fluoride gel in serum was bactericidal for the tested species after 10 min contact time, with exception of F. nucleatum.

Actinobacillus↗

PubMed1997

Studies on diffusion in alginate gels. II. Effect of acid and subsequent re-exposure to calcium on the diffusion of caffeine and theophylline in alginate gel films.

The aim of this research was to investigate the effect of acid exposure and calcium re-exposure on the diffusion of caffeine and theophylline through calcium alginate gel films. Diffusion was measured using side-by-side glass cells, before and after the films were exposed to simulated gastric fluid (SGF) USP minus pepsin. The permeability increased by about 15-fold for theophylline and 48-fold for caffeine after 5 min SGF exposure. There was no significant further increase by prolonging the exposure time to 4 hr. The diffusion of both drugs was less than through alginate gel films that were not cross-linked with calcium but were gelled in SGF. Treatment of the calcium alginate gel films with SGF depleted the films of all of their calcium content. Calcium alginate gel films that were exposed to SGF for a constant period of 15 min, were re-exposed to 0.34 M calcium chloride solution for periods up to 24 hr. The calcium content returned to 80% of the level in the original calcium alginate films and diffusion was reduced, but not to the level that was observed in the original calcium alginate films. It was concluded that the effect of SGF on calcium alginate was very rapid and that the increase in drug diffusion observed was probably due to removal of calcium from the gel. It was also concluded that the original diffusion properties and calcium content could only be partially regenerated by re-exposing the acid-exposed films to calcium chloride.

Administration, Oral↗

PubMed1988

"High-resolution" mini-two-dimensional gel electrophoresis automatically run and stained in less than 6 h with small, ready-to-use slab gels.

Although two-dimensional (2-D) gel electrophoresis is one of the most powerful techniques for analyzing protein mixtures, its application in routine clinical laboratories is currently limited, because it is time-consuming, complex, and relatively expensive. Here we describe a method for automatically running and staining "high-resolution" mini 2-D electrophoresis gels in less than 6 h, by using "ready-to-use" slab gels and a PhastSystem electrophoresis apparatus. We present 2-D gel electrophoretograms of 25 nL of plasma, as well as their automatic computer analysis. For comparison, a conventional 2-D gel electrophoresis profile of 200 nL of a plasma sample is shown. The technique is easy to perform, highly sensitive, rapid, and potentially useful in semi-routine clinical chemistry laboratories.

Autoanalysis↗

PubMed1992

Separation of oligogalacturonic acids by high-performance gel filtration chromatography on silica gel with diol radical.

Oligogalacturonic acids (OLGAs) ranging from two to nineteen residues in length were separated using high-performance gel filtration chromatography on a silica gel with diol radical. The optimum conditions (eluent, column temperature) for separation of OLGAs by high-performance gel filtration chromatography were investigated. The column used in this experiment allowed a high pressure of 4900 p.s.i. and a flow-rate of 2 ml/min. The stationary phase of silica gel stabilized the separation of OLGAs. The peaks of OLGAs separated using this column were assigned by comparing retention times with standards, and the molecular weights of the corresponding OLGAs were determined by fast atom bombardment mass spectrometry.

Chromatography, Gel↗

PubMed1989

Studies of BCR and ABL gene rearrangements in chronic myelogenous leukemia patients by conventional and pulsed-field gel electrophoresis using gel inserts.

Continual monitoring of the presence of the Philadelphia (Ph) chromosome in patients with chronic myelogenous leukemia (CML) is important for diagnosis as well as evaluation of therapy response of these patients. Because the Ph chromosome has been characterized molecularly to involve a reciprocal translocation between the ABL and BCR genes, there is an increasing interest in the use of molecular probes to detect chromosomal rearrangements in this disease. While rearrangements involving the bcr region of the BCR gene can be detected by conventional gel electrophoresis (CGE), detection of those involving ABL generally requires pulsed-field gel electrophoresis (PFGE). Currently, however, CGE and PFGE require different methods of cell preparation, with isolated DNA used in CGE and gel inserts containing whole cells used in PFGE. In this study, we show that the gel-insert method of DNA preparation can be adapted for use in CGE with slight modification of the gel-running conditions. The advantages of this method are demonstrated by studying both bcr and ABL rearrangements in bone marrow and peripheral blood samples of CML patients. Furthermore, we report a novel finding that chromosomal breakpoints in the ABL gene of CML patients occur predominantly between exons 1b and 1a.

Bone Marrow↗

PubMed1980

Concentration-dependent migration in gel chromatography of reversibly polymerizing solutes: the osmotic behavior of polyacrylamide gels.

The osmotic behavior of gels derived from polyacrylamide has been measured in order to establish the validity of assuming linear concentration dependence of bead shrinkage upon osmotic pressure in quantitative studies of reversibly associating solutes by gel chromatography on Bio-Gel. The concentration dependence of elution volume predicted with due allowance for this osmotic shrinkage yields theoretical curves that provide good descriptions of experimental results obtained in chromatography of alpha-chymotrypsin on Bio-Gel P-30 equilibrated with acetate-chloride buffer, pH 3-86, 10.20; and also of bacterial alpha-amylase on Bio-Gel P-150 equilibrated with 0.10 M NaCl-0.015 M calcium acetate-0.010 M EDTA, pH 7.0. For the former system the osmotic effect has negligible consequences on the quantitative interpretation of the results. With the alpha-amylase system, however, consideration of the osmotic effect is necessary to obtain even a qualitative indication of the existence of the monomer-dimer equilibrium.

Journal Article↗

PubMed2000

A simple method for non-radioactive PCR-SSCP using MDE gel solution and a midi gel format: application for the detection of variants in the GLUT1 and CTLA-4 genes.

The need to identify disease-causing mutations and DNA polymorphisms has increased with the continuing identification of new candidate genes. PCR single-strand conformation polymorphism (PCR-SSCP) is one of the techniques most widely used to identify a mutant sequence or a polymorphism in a known gene. However, the original SSCP protocols using the incorporation of radioactive label and polyacrylamide gel electrophoresis on sequencing gels for detection were labour intensive and time-consuming. Here we describe a simple SSCP protocol using MDE gel solution and a midi gel format to detect SSCP variations in the glucose transporter gene GLUT1, that we have previously analysed with the standard radioactive SSCP protocol, and we have also tested this method on the previously described point mutation (A/G transition in exon 1) of the CTLA-4 (cytotoxic T lymphocyte associated-4) gene. All known variants were detected. Based on the results, this technique appears to be simple, with no use of radioactive labels and with easy handling of the gel. Furthermore, it needs little optimisation, is relatively rapid and highly sensitive. We propose this method for the first screening for candidate gene variants.

Abatacept↗

PubMed1997

Osmotic stress induces a phase transition from interdigitated gel phase to bilayer gel phase in multilamellar vesicles of dihexadecylphosphatidylcholine.

We have investigated the effects of poly(ethylene glycol) (PEG) on the structure and phase behavior of multilamellar vesicles of dihexadecylphosphatidylcholine (DHPC-MLVs) using an X-ray diffraction method. At low concentrations of PEG-6K (MW = 7500), DHPC-MLVs were in an interdigitated gel (L(beta)I) phase, a gel phase with interdigitated hydrocarbon chains. At around 24% (w/v) PEG 6K, a phase transition from the L(beta)I phase to a bilayer gel phase occurred in the DHPC-MLVs, and above this concentration, they were in a bilayer gel phase. On the other hand, ethylene glycol (EG), the monomer of PEG, did not induce this phase transition in the DHPC-MLVs. A mechanism of this phase transition is proposed and discussed; a decrease in the repulsive interaction between the head groups of the phospholipids in the bilayer gel phase with an increase in PEG concentration, which is due to a decrease in the cross-sectional area of the head group region by osmotic stress, may be the main reason for this phase transition.

Journal Article↗

PubMed2005

Dispersion of acid-treated carbon nanofibers into gel matrices prepared by the sol-gel method.

1-Naphthol has been used as an in-situ fluorescent probe to characterize the dispersibility of carbon nanofibers (CNFs) into the sol-gel matrix of silicon alkoxide. The ion-pair fluorescence of 1-naphthol was found in the gel dispersing acid-treated CNFs instead of 1Lb fluorescence, which was preferred in the low polar gel matrix. This indicates that 1-naphthol easily interacts with oxidized groups present on the surface of the acid-treated CNFs due to the high dispersibility of the CNFs into the gel matrix. The oxidized groups on the CNF surface are useful for preventing self-assembly and/or aggregation of the CNFs in the gel matrix.

Journal Article↗

PubMed2004

Concentration fluctuations in polymer gel investigated by neutron scattering: static inhomogeneity in swollen gel.

By using small-angle neutron scattering (SANS) and neutron spin echo (NSE), we have quantitatively investigated the static inhomogeneity in poly (N-isopropyl acrylamide) gel (PNIPA) in microscopic length scales of 0.015<q<0.16 A(-1), where q is a wave number of scattered neutrons. NSE revealed that at lower q( congruent with 0.015 A(-1)), the concentration fluctuations in the PNIPA gel decays more slowly as compared to the PNIPA solution without crosslinks. According to our scenario that the slower decay found for the PNIPA gel is due to the static inhomogeneity coexisting in the swollen gel, small-angle scattering S(q) obtained by SANS has been quantitatively decomposed into thermal and static scattering components, respectively, S(th)(q) and S(st)(q). It was further revealed that (i) the q-region where S(st)(q) becomes dominant is closely related to that for the abnormal butterfly scattering under stretching, and (ii) as the temperature increases toward the temperature for volume phase transition, S(st)(q) of a squared Lorentzian shape increases more drastically than S(th)(q) of a Lorentzian shape. These findings were quantitatively understood in the theoretical framework by Panyukov and Rabin [Macromolecules 29, 7960 (1996)] or by Onuki [J. Phys. II. France 2, 45 (1992)], taking into account stress-fluctuation coupling under coexistence of the inherent structural heterogeneity in the real gel. We further found that the static inhomogeneity showing S(st)(q) seems to relate to the necklacelike microstructure, appearing after a shallow quench into the collapsed phase.

Journal Article↗

PubMed1967

Agar-gel precipitin-inhibition test for coccidioidomycosis. I. Preliminary evaluation of the complement-fixation and agar-gel precipitin tests in the serodiagnosis of human coccidioidomycosis.

The agar-gel precipitin-inhibition serological test for coccidioidomycosis was a more sensitive indicator of Coccidioides immitis antibodies than the tube precipitin, the agar-gel immunodiffusion, in the complement-fixation tests in assaying monkey sera, whether these sera were from prechallenge-vaccinated or postchallenged animals. When applying this technique to the assay of human sera, an analogous finding generally persisted. However, some human sera were positive by the complement-fixation test and negative by the agar-gel precipitin-inhibition test. These sera were diffused in agar-gel against various coccidioidin complement-fixation, tube precipitin, and agar-gel precipitin-inhibition test antigens with essentially negative results.

Animals↗

PubMed2002

DNA electrophoresis in agarose gels: effects of field and gel concentration on the exponential dependence of reciprocal mobility on DNA length.

Electrophoretic mobilities of DNA molecules ranging in length from 200 to 48 502 base pairs (bp) were measured in agarose gels with concentrations T = 0.5% to 1.3% at electric fields from E = 0.71 to 5.0 V/cm. This broad data set determines a range of conditions over which the new interpolation equation nu(L) = (beta+alpha(1+exp(-L/gamma))(-1) can be used to relate mobility to length with high accuracy. Mobility data were fit with chi(2) > 0.999 for all gel concentrations and fields ranging from 2.5 to 5 V/cm, and for lower fields at low gel concentrations. Analyses using so-called reptation plots (Rousseau, J., Drouin, G., Slater, G. W., Phys. Rev. Lett. 1997, 79, 1945-1948) indicate that this simple exponential relation is obeyed well when there is a smooth transition from the Ogston sieving regime to the reptation regime with increasing DNA length. Deviations from this equation occur when DNA migration is hindered, apparently by entropic-trapping, which is favored at low fields and high gel concentrations in the ranges examined.

DNA↗

PubMed1988

Rapid imaging, using a cooled charge-coupled-device, of fluorescent two-dimensional polyacrylamide gels produced by labelling proteins in the first-dimensional isoelectric focusing gel with the fluorophore 2-methoxy-2,4-diphenyl-3(2H)furanone.

A new method for visualising proteins in two-dimensional polyacrylamide gels was developed. Proteins were labelled with the fluorophore 2-methoxy-2,4-diphenyl-3(2H)furanone (MDPF) while present in the first-dimensional gel after isoelectric focusing and subsequently electrophoresed into the second-dimensional gel. High resolution spot patterns were produced and compared with other methods of visualisation. A new rapid imaging system based on a cooled charge-coupled-device was used to view the two-dimensional fluorescent protein spot patterns. The method allows the immediate and rapid imaging of two-dimensional gels at the end of electrophoresis with no further processing.

Bacterial Proteins↗

PubMed1990

Fabric-reinforced ultrathin polyacrylamide gels for sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing.

The suitability of four different fabric materials for the preparation of ultrathin rein-forced polyacrylamide gels was investigated. With all fabric-reinforced gels, a good separation of proteins by isoelectric focusing and sodium dodecyl sulfate-electrophoresis could be achieved. Semi-dry electrophoretic blotting of proteins was possible with all types of fabric-reinforced gels. Two polyester fabrics (a net and a fleece) were decidedly superior in handling and dimensional stability on drying to a nylon fabric and another polyester fleece material. Only gels prepared with the former materials withstood further treatment, such as fixation, staining, destaining, and drying. One of the polyester fleece fabrics had poor handling properties and the nylon fabric was unsuitable for direct staining procedures employing concentrated (20% w/v) trichloroacetic acid as fixative.

Electrophoresis, Polyacrylamide Gel↗

PubMed1993

Apolipoprotein (a): a comparison of isoforms identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or by sodium dodecyl sulfate-agarose gel electrophoresis.

Lipoprotein(a) resembles low density lipoprotein in structure, except that a unique apolipoprotein (apo), apo(a), is linked to apo B-100. Variations in the number of sequence repeats in the apo(a) gene give rise to a range of isoforms. Depending on the method used, 6-30 apo(a) isoforms have been observed; however, the correspondence of these different isoforms has not been reported, making between-study comparisons difficult. In the present study we address this question by characterizing the apo(a) phenotypes of 48 sera using two previously reported separation methods, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, 3-12% gels) and SDS-agarose gel electrophoresis. In addition, the molecular weight of each isoform was estimated using haptoglobin 2-2 polymers as molecular weight standards. Among the 48 sera, 15 distinct apo(a) isoforms were separated by SDS-PAGE and 28 by SDS-agarose gel electrophoresis. There was excellent correlation between the two nomenclature systems (r = -0.97, p < 0.001, by rank correlation), and the ranges were totally overlapping, with the same two isoforms being identified as the largest and smallest by either method. The apparent molecular mass range for the isoforms was 294-624 kDa, which is in close agreement with the theoretical molecular mass range of 238-643 kDa, calculated from the sequence and carbohydrate content of recombinant apo(a). The disparity in number of isoforms between methods was expected, due to the poorer separation of apo(a) by SDS-PAGE; 3.1 +/- 1.7 (median, 2.0) SDS-agarose isoforms were combined for each SDS-PAGE isoform.(ABSTRACT TRUNCATED AT 250 WORDS)

Apolipoproteins A↗

PubMed1998

Agarose gel structure using atomic force microscopy: gel concentration and ionic strength effects.

Agarose gels have been studied by atomic force microscopy (AFM). The experiments were especially designed to work in aqueous conditions, allowing direct observation of the "unperturbed" gel without invasive treatment. AFM images clearly show strong dependence of pore diameter and its distribution on ionic strength of the solvent. As the ionic strength increases, the distribution becomes broader and the position of its maximum shifts toward higher values. The evolution of the distribution curves indicates that gels become more homogeneous with decreasing Tris-borate-EDTA (TBE) buffer concentration. An empirical law of the mean pore diameter as a function of the ionic strength is established. In agreement with our previous work we found that, for a given ionic strength, the pore diameter increases when the agarose concentration decreases and that the wide pore diameter distribution narrows as the gel concentration increases.

Electrophoresis, Agar Gel↗

PubMed1987

Molecular weight standards for calibration of gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis: ferritin and apoferritin.

Ferritin and apoferritin are widely used for the calibration of gel filtration columns and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and are commercially offered for these purposes as part of molecular weight calibration kits. Many of the reported applications are severely in error as presented in leading references and application manuals. The manufacturers have based their recommendations on incorrect physicochemical parameters in the literature and incorrect or inadmissible assumptions about the protein subunit composition and architecture and have not taken into account the unusual resistance of these proteins to denaturation in SDS. Here the relevant physicochemical parameters of horse spleen apoferritin as reported in the literature are critically reevaluated and the best current estimates are identified as the following: weight average molecular weight of apoferritin, Mw = 481,200; molecular weight of subunits, major subunit, ML = 19,889; minor subunit, MH = 22,200; apparent specific volumes in 0.02 M acetate buffer, pH 5.5, and 0.1 M NaCl, phi = 0.721 ml g-1 and phi' = 0.743 ml g-1; partial specific volume at 20 degrees C, v = 0.738 ml g-1; viscosimetric molar volume, M[n] = 1.78 X 10(6) ml mol-1; Stokes radius, RSt = 67.1 A; viscosimetric radius, Rvis = 65.6 A; sedimentation coefficient S degrees 20, w = 16.6 S; translational diffusion coefficient, D20, w = 3.24 X 10(-7) cm2 s-1. Recommendations are provided for proper application of ferritin and apoferritin for calibration purposes in gel filtration and SDS-polyacrylamide gel electrophoresis.

Animals↗

PubMed1975

Agarderivatives for chromatography, electrophoresis and gel-bound enzymes. IV. Benzylated dibromopropanol cross-linked sepharose as an amphophilic gel for hydrophobic salting-out chromatography of enzymes with special emphasis on denaturing risks.

The preparation of benzylated covalently cross-linked Sepharose 2B is described. Such gel was analyzed for its degree of substitution, and gels with three different degrees of substitution were used in chromatographic experiments with dextranase, alpha-amylase, lactate dehydrogenase, alpha-chymotrypsin and trypsin. Yields and chromatographic patterns for different eluting systems were determined. It was found that gradients combining an increase in ethylene glycol concentration with a decrease in salt concentration gave better results than did pure salt gradients. No denaturation was observed for dextranase or alpha-amylase, but the other enzymes tested were partly denatured. The most severe denaturation was observed for lactate dehydrogenase desorbed from the highest substituted gels, although the enzyme was highly active in the adsorbed state. The results and the use of amphophilic gels are discussed.

Agar↗