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PubMed2002

In-gel deglycosylation of sodiumdodecyl sulfate polyacrylamide gel electrophoresis-separated glycoproteins for carbohydrate estimation by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Mass determination by mass spectrometric methods (electrospray ionization mass spectrometry (ESI-MS), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS)) of sodiumdodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)-separated proteins is a well known procedure and reliable protocols are available. In our efforts to use the established methods to determine the molecular mass of the disulfide bridged, heterodimeric glycoprotein GP3 and to determine the carbohydrate content of each protein subunit we developed an in-gel chemical deglycosylation method. For this purpose we established experimental conditions that allow maximum extraction of the high molecular mass protein subunits and developed a routine method to apply the HF-pyridine deglycosylation protocol to proteins isolated from polyacrylamide gel pieces. The novel protocol and extraction procedure described can be used to analyze O-glycosylated proteins up to 150 kDa after SDS-PAGE separation.

Animals↗

PubMed2005

Improvements in two-dimensional gel electrophoresis by utilizing a low cost "in-house" neutral pH sodium dodecyl sulfate-polyacrylamide gel electrophoresis system.

Two-dimensional gel electrophoresis (2-DE) is widely used for initial protein separation in proteomics. Commercial products using neutral pH sodium dodecyl sulfate-polyacrylamide gel electrophoresis ((SDS-PAGE)/(Bis (2-hydroxyethyl) imino-tris (hydroxymethyl) methane-HCl, or Bis-Tris)) have greatly improved this technique, but cost and limited sizes restrict their applications. An "in-house" system is presented, resulting in better resolution, separation, and new spot visualization and improved resolution when compared to Tris-HCl gels. Their utility is demonstrated using albumin-depleted serum samples, rabbit heart left ventricle, and human immunodeficiency virus type 1 (HIV-1).

Animals↗

PubMed1994

Comparison of the separation of proteins by sodium dodecyl sulfate-slab gel electrophoresis and capillary sodium dodecyl sulfate-gel electrophoresis.

Capillary sodium dodecyl sulfate-gel electrophoresis, a one-dimensional version of the well-established planar analytical method of SDS-polyacrylamide gel electrophoresis, has proven a powerful new microanalytical method for the separation of protein molecules according to their size. In this paper the planar SDS-PAGE and the capillary SDS-gel electrophoresis of different proteins are compared with respect to performance, precision, and efficiency. Molecular weight versus migration distance and migration time curves for 65 proteins, running from 14,200 to 205,000 Da, are given for both methods.

Capillary Action↗

PubMedDate not supplied

Randomized comparison of two nystatin oral gels with miconazole oral gel for treatment of oral thrush in infants. Antimycotics Study Group.

Recently, two commercial oral nystatin gels have been marketed in Germany (Candio-Hermal, Hermal; Lederlind, Lederle). In a prospective open randomized multicentre study involving 12 paediatricians in private practice, Candio-Hermal and Lederlind were compared with miconazole oral gel (Daktar, Janssen) in 95 infants with oral thrush (candidosis). Oral and rectal swabs were taken on days 0 and 14. Treatment duration varied according to the amount of drug contained in one tube (Candio-Hermal 10 days; Lederlind 14 days; Daktar 8 days). On day 14, clinical cure was observed in 23 of 27 infants treated with Daktar (85.1%), in 15 of 35 infants treated with Candio-Hermal (42.8%; p < 0.0007); and in 16 of 33 infants treated with Lederlind (48.5%; p < 0.004). Clinical relapses were observed in 15 patients (Candio-Hermal: n = 9; Lederlind: n = 6). The overall oral mycologic cure rate was significantly higher with Daktar (29.6%) and Candio-Hermal (20.0%) than with Lederlind (3.0%; p < 0.005 and < 0.03, respectively). It can be concluded that Daktar oral gel is significantly more effective than Candio-Hermal and Lederlind in curing oral thrush in infants.

Candidiasis, Oral↗

PubMed1995

Labor induction with intravaginal misoprostol versus intracervical prostaglandin E2 gel (Prepidil gel): randomized comparison.

OBJECTIVE: Our purpose was to compare the safety and efficacy of intravaginal prostaglandin E1, misoprostol, with that of intracervical prostaglandin E2 (Prepidil gel) for labor induction. STUDY DESIGN: One hundred three patients with an indication for induction of labor were randomly assigned to induction with prostaglandin E1, 50 micrograms intravaginally, or with Prepidil gel, 0.5 mg intracervically, every 4 hours until active labor. RESULTS: Four patients were excluded, leaving 49 patients who received prostaglandin E1 and 50 who received prostaglandin E2. The time from start of induction to vaginal delivery was significantly shorter in the prostaglandin E1 group (11.4 vs 18.9 hours, p < 0.001), and fewer patients in the prostaglandin E1 group required oxytocin augmentation (23% vs 55%, p < 0.005). No significant differences were noted in mode of delivery or in adverse maternal, fetal, or neonatal effects. CONCLUSION: Intravaginal prostaglandin E1 is a more effective, lower-cost agent for induction of labor than is intracervical prostaglandin E2 gel and is comparable in safety.

Administration, Intravaginal↗

PubMed1978

Preparative gel electrophoresis: detection, excision, and elution of protein bands from unstained gels.

Methods are described for localizing proteins in unstained gels and accurately excising the regions containing them. A gel elutor, which is all glass with Lucite fittings, is also described. The elutor removes proteins from gels with a high yield and concentrates the protein in a small volume. The elutor is simple and very easy to use. A way is presented for avoiding the oxidation of methionine and cysteine during preparative electrophoresis.

Acetic Acid↗

PubMed1978

Direct physical measurements on substituted agarose gels: evidence for intercalation of gel-bound ethidium into transfer RNA.

The cation of the salt ethidium bromide (3,8-diamino-5-ethyl-6-phenylphenanthridinium bromide) has been covalently linked to an agarose matrix through an intermediate 3,3'-diaminodipropylaminosuccinyl spacer arm. Partition binding and visible absorption spectral measurements on the gel were used to monitor the binding of transfer RNA to the covalently bound ethidium group. Direct fluorescence measurements of the formation of the gel-bound complex indicate that this binding involves the intercalation of the ethidium groups into the tRNA molecule. Dissociation of the ethidium-tRNA complex was monitored as a function of sodium chloride concentration by both direct solution spectral measurement of the released tRNA and by fluorescence quenching measurements of the dissociation of the intercalation complex. The derivatized gel has been shown to be capable of the fractionation of tRNA species by elution with a positive salt gradient under column flow conditions.

Chromatography, Affinity↗

PubMed1986

A method for the direct measurement of glycogen synthase activity on gels after polyacrylamide gel electrophoresis.

A method for the detection of glycogen synthase activity after nondenaturing polyacrylamide gel electrophoresis is described. After the electrophoretical run, the gels were incubated in situ with UDP-glucose and glycogen. Labeled or unlabeled UDP-glucose could be used, since similar activity patterns were obtained by autoradiography or iodine staining of the gels. The method here described offers several advantages in terms of speed, sensitivity, and economy when compared with other procedures.

Animals↗

PubMed1988

Mixed anionic detergent/aliphatic alcohol-polyacrylamide gel electrophoresis alters the separation of proteins relative to conventional sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

The order and relative mobility of proteins on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is affected by unknown components that are differentially present in SDS preparations obtained from different sources [J.B. Swaney, G.F. Vande Woude, and H.L. Bachrach (1974) Anal. Biochem. 58, 337-346]. The modified separation capabilities of such SDS preparations are useful but the use of this phenomenon in a controlled manner requires that the components responsible for the altered separation be identified. Accordingly, this paper describes a polyacrylamide gel electrophoresis system [mixed alcohol/detergent-polyacrylamide gel electrophoresis (MAD-PAGE)] that employs a mixture of alcohol and detergent instead of SDS alone to modify and enhance protein separation relative to conventional SDS-PAGE. A defined mixture consisting of four sulfated alkyl detergents (dodecyl sulfate, tetradecyl sulfate, hexadecyl sulfate, octadecyl sulfate) as well as the four alcohols of corresponding aliphatic chain length was found to be effective at duplicating the electrophoretic effect of USP-grade SDS and thus changed the relative order and position of polypeptides on electrophoresis relative to conventional SDS-PAGE. This method serves as an adjunct to conventional SDS-PAGE by providing another means of resolving proteins that are not normally resolved by SDS-PAGE. Further, it was found that MAD-PAGE is capable of resolving the NS1 protein of influenza virus into three fractions, whereas conventional SDS-PAGE yields one electrophoretic species. Reelectrophoresis of these novel NS1 bands by conventional SDS-PAGE indicated that they were not modified during MAD-PAGE and probably represented distinct molecular forms present in infected cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Alcohols↗

PubMed1981

Studies on the special properties of actomyosin in the gel form. II. An analysis of the turbidity changes seen in gel suspensions.

The turbidity changes induced by MgATP in suspensions of actomyosin gel particles have been studied systematically over a wide range of MgATP concentrations at different temperatures with and without the regulatory proteins. An analysis of these changes distinguishes three separate protein-protein interactions in the gel: (1) The transient cyclic interactions between actin and myosin involved in the hydrolysis of MgATP and contraction. (2) Cross-links that cause turbidity in the original suspension. (3) Cross-links that cause the high turbidity usually associated with superprecipitation. In general, there was a good correlation between the half-time for reaching maximum turbidity during superprecipitation and the rate of hydrolysis. On the other hand, the actual magnitude of the turbidity increase was progressively diminished as the concentration of substrate was raised in the millimolar range. It appears that some essential phase of the superprecipitation process is limited by the same enzymatic step that limits the rate of hydrolysis. However, whether or not this phase leads to an increase in turbidity depends on the concentration of MgATP. Apparently, high physiological levels of MgATP (1-5 mM) inhibit the formation of the specific cross-links that cause high turbidity in the superprecipitate. It is proposed that MgATP interferes with these links when it can bind to a low-affinity site on myosin that is separate from the high-affinity active sites for hydrolysis. Thus, in contracting muscle, interfilament interactions analogous to those that increase turbidity and cause isodimensional shrinkage in the gel would be prevented by the high level of MgATP in the sarcoplasm. Observations relating the shortening of isolated myofibrils to their turbidity in suspension lend support to this interpretation.

Actomyosin↗

PubMed1986

Ripening of the cervix with prostaglandin E2-gel. A randomized study with a new ready-to-use compound of triacetin-prostaglandin-E2-gel.

A randomized study with a group of patients treated with a new ready-to-use triacetin-prostaglandin E2-gel compared to a non-treated group was conducted. The gel-treated group showed a distinct difference in cervical score after 12 h and ten patients were delivered during this period without further induction attempt compared to none in the control group. There was a significantly lower need for oxytocin stimulation in the treated group (P less than 0.0005), but there was no difference in the cesarean section rate or instrumental delivery rate. No side-effects were seen. This new gel seems effective and safe.

Adult↗

PubMed1993

Affinity gel electrophoresis of nucleic acids. Nucleobase-selective separation of DNA and RNA on agarose-poly(9-vinyladenine) conjugated gel.

Poly(9-vinyladenine) (PVAd) was immobilized within an agarose gel matrix to produce a novel affinity gel for the base-specific separation of nucleic acids by electrophoresis. The shape (single- or double-stranded) and base content of nucleic acids were specifically recognized by the affinity gel. Only single stranded DNA, of which the sequence is not regular enough to form a stable duplex hairpin structure, was selectively absorbed over double-stranded DNA. Among five polynucleotides having different bases such as poly(A), poly(G), poly(C), poly(U) and poly(I), poly(U) and poly(I) were base-specifically adsorbed by PVAd, probably by hydrogen bond formation. The effect of the molecular mass and size of poly(9-vinyladenine) was also examined.

Adenine↗

PubMed2004

Gel-to-gel phase transition may occur in mammalian cells: Mechanism of formation of "dark" (compacted) neurons.

In the course of many diseases, individual non-apoptotic cells that are randomly distributed among undamaged cells in various mammalian tissues become shrunken and hyperbasophilic ("dark"). The light microscopic shrinkage is caused by a potentially reversible, dramatic compaction of all ultrastructural elements inside the affected cells, and escape of the excess water through apparently intact plasma membrane. In the case of neurons, the ultrastructural compaction rapidly involves the soma-dendrite domains in an all-or-nothing manner, and also mm-long axon segments. The present paper demonstrates that such ultrastructural compaction in neurons, which affects the whole soma-dendrite domain or long axon segments, can take place both immediately after an in vivo head injury and in rat brains perfusion-fixed for 30 min., and then chilled to just above the freezing point before the same kind of head injury was inflicted. This argues strongly against any enzyme-mediated compaction mechanism. On the analogy of gel-to-gel phase transitions in polymer chemistry, we hypothesize a pure physico-chemical compaction mechanism. Specifically, after initiation at a single site in each affected cell, the ultrastructural compaction is propelled throughout the whole cell on the domino principle by the free energy stored in the form of non-covalent interactions among the constituents of some cytoplasmic gel structure.

Animals↗

PubMed2005

Denaturing gradient gel electrophoresis gel expansion (DGGEGE)--an attempt to resolve the limitations of co-migration in the DGGE of complex polymicrobial communities.

Recent molecular approaches for the study of microbial communities such as PCR-cloning have enabled the detection and identification of as-yet-unculturable taxa. Cloning and sequencing of multiple samples is extremely laborious and expensive to perform thoroughly due to the large diversity involved. For this purpose, techniques such as denaturing gradient gel electrophoresis (DGGE) may be better suited. There is increasing evidence suggesting that DGGE of complex polymicrobial communities may be limited by co-migration of different sequences. In this study, we attempt to address this limitation by excising individual bands and running them through a shorter denaturant gradient, a process we have termed "denaturing gradient gel electrophoresis gel expansion" (DGGEGE).

Bacteria↗

PubMed2005

A randomized, double-blind, vehicle-controlled, bilateral comparison trial of bexarotene gel 1% versus vehicle gel in combination with narrowband UVB phototherapy for moderate to severe psoriasis vulgaris.

We report results of a randomized, vehicle-controlled, bilateral comparison pilot study of bexarotene gel 1% with narrowband UVB (NBUVB) phototherapy for moderate to severe psoriasis. In all, 9 patients applied drug or vehicle gel to comparable target lesions up to twice daily for 10 weeks. NBUVB was initiated 2 weeks after topical therapy began. Limitations include small sample size and interim analysis. Based on analysis of target lesion scores, bexarotene gel 1%/NBUVB was significantly more effective than placebo/NBUVB.

Administration, Topical↗

PubMed1997

A random-walk model for retardation of interacting species during gel electrophoresis: implications for gel-shift assays.

We recently showed that intermolecular DNA triplexes can form during gel electrophoresis when a faster migrating single strand overtakes a slower migrating band containing a duplex of appropriate sequence. We proposed a model to account for the resulting apparent comigration of triplexes with the duplex band when the lifetime of the triplex is much shorter than the time of electrophoresis. The model predicts that short-lived complexes can be detected by a gel-shift assay if the faster migrating component of the complex is labeled, a slower migrating component is in excess, and the complex itself migrates more slowly than either of the components. In this case the labeled component, after dissociation from the complex, overtakes a slower migrating band of the free, unlabeled second component and can be captured by the unlabeled component and again retarded; after dissociation of the newly formed complex the cycle is repeated. If the concentration of unlabeled component in the band is larger than some critical value (c(cr)), most of the labeled component becomes trapped in this band during the entire time of gel electrophoresis, thus effectively comigrating with the slower migrating unlabeled component. We call this mechanism of comigration "cyclic capture and dissociation" (CCD). Here we present a quantitative analysis of the model of CCD comigration which predicts that CCD comigration can be used not only for the detection of relatively short-lived complexes, but also for estimation of the specificity of complex formation.

Base Composition↗

PubMed1999

Weekly administration of prostaglandin E2 gel compared with expectant management in women with previous cesareans. Prepidil Gel Study Group.

OBJECTIVE: To compare the clinical effectiveness and safety of outpatient administration of intracervical prostaglandin (PG) E2 gel with expectant treatment for women desiring vaginal births after cesareans. METHODS: This was a randomized, multicenter investigation involving term pregnant women who each had one previous low-transverse cesarean and an unfavorable cervix (Bishop score no more than 6), and who was a candidate for vaginal delivery. They were assigned to receive 0.5 mg of PGE2, (Prepidil; Pharmacia-Upjohn, Kalamazoo, MI) intracervically at 39 weeks' gestation, repeated at weekly office visits for up to three doses, or expectant treatment. The main outcome variable was vaginal birth. RESULTS: Of 294 cases, 143 received gel and 151 were treated expectantly. No differences between groups were found for maternal age, race, or Bishop score. Compared with the expectant treatment group, the PGE2 gel group was not more likely to deliver sooner or vaginally (57% versus 55%, P = .68). The onset of labor, duration of labor among those delivering vaginally, and 1- and 5-minute Apgar scores were not different between groups. No uterine ruptures occurred, and adverse effects were equally likely in both groups. CONCLUSION: Although its safety was confirmed for outpatient use, weekly doses of intracervical PGE2 did not improve the likelihood of vaginal births after cesareans.

Adolescent↗

PubMed2002

Osteoblast differentiation of bone marrow stromal cells cultured on silica gel and sol-gel-derived titania.

Primary cultures of osteogenic precursor cells derived from rat bone marrow stroma were performed on commercially available pure titanium discs (Ti c.p.) and surface modified Ti c.p.using a sol-gel technique (Ti sol). In separate repeated experimental runs, cell behavior and in vitro mineralization were compared with cultures on silica gel bioactive glass discs (S53P4). All substrates were incubated in simulated body fluid prior to the experiment. Overall, variable effects between experimental runs were seen. Apparently, this was due to the heterogeneous nature of the used cell population. Therefore, only careful conclusions can be made. Initial cell adhesion and growth rates between 3 and 5 days of culture--analyzed by cell numbers--were in general comparable for the two titanium substrates, while initial growth up to day 3 is suggested to be higher in Ti c.p. compared to Ti sol. Although initial cell adhesion on the S53P4 glass discs was lower than the titanium substrates, cell growth rates appeared to be higher on the silica gel compared to the two titanium substrates. Further, there were some indications that the early and late osteoblast differentiation markers, alkaline phosphatase and osteocalcin, monitored up to day 24, were elevated in Ti c.p cultures compared to Ti sol cultures. There were no differences observed in in vitro mineralization between the titanium groups. S53P4 seemed to display a substantially higher differentiating capacity for both osteogenic cell markers as well as in vitro mineralization compared to the two titanium substrates.

Alkaline Phosphatase↗