Biomedical subjects

M A Strausbauch

Publications and source records attributed to M A Strausbauch.

10 recordsLinked to original sources

PubMed1999

Temporal sequence of transcription of perforin, Fas ligand, and tumor necrosis factor-alpha genes in rejecting skin allografts.

BACKGROUND: Perforin, Fas ligand (FasL), and tumor necrosis factor-alpha (TNF-alpha) have been implicated in cytolytic T lymphocyte (CTL) effector function. However, the relative roles of these effector molecules in allograft rejection are unclear, and there has been no rigorous quantitation of transcription of the respective genes throughout the period from transplantation to rejection. METHODS: We orthotopically transplanted mouse tail skin allografts and estimated the numbers of transcripts of these genes expressed by graft-infiltrating T cells with rigorous quantitative, competitive reverse transcribed PCR (QC-RT-PCR) that enabled the comparison of transcription of different genes. RESULTS: Perforin and FasL mRNA levels correlated closely with the rejection of allografts by normal hosts over the 4 days preceding rejection. Antibody-mediated depletion of host CD4+ T cells retarded perforin transcription and significantly suppressed FasL transcription, suggesting FasL was not required for allograft rejection. TNF-alpha transcription was the highest of these genes in this time period, but these levels were dwarfed by TNF-alpha transcription at 24 hr posttransplant when transcription in both autografts and allografts was 30-fold higher than in allografts on the day before rejection. Elimination of the function of these single or paired genes through genetic mutation or antibody treatment had no significant effect on the speed of rejection. CONCLUSIONS: The levels of perforin and FasL transcription appeared to be related to the process of allograft rejection in normal hosts. However, TNF-alpha transcription was highest during the posttransplant period suggesting that the principal role of TNF-alpha is in wound-healing rather than the effector phase of rejection.

Animals↗

PubMed1998

Infrared-mediated thermocycling for ultrafast polymerase chain reaction amplification of DNA.

Interest in improving the speed of DNA analysis via capillary electrophoresis has led to efforts to integrate DNA amplification into microfabricated devices. This has been difficult to achieve since the thermocycling required for effective polymerase chain reaction (PCR) is dependent on an effective contact between the heating source and the PCR mixture vessel. We describe a noncontact method for rapid and effective thermocycling of PCR mixtures in electrophoretic chip-like glass chambers. The thermocycling is mediated through the use of a tungsten lamp as an inexpensive infrared radiation source, with cooling effected with a solenoid-gated compressed air source. With temperature ramping between 94 and 55 degrees C executed in glass microchambers as rapidly as 10 degrees C/s (heating) and 20 degrees C/s (cooling), cycle times as fast as 17 s could be achieved. Successful genomic DNA amplification was carried out with primers specific for the beta-chain of the T-cell receptor, and detectable product could be generated in a fraction of the time required with commercial PCR instrumentation. The noncontact-mediated thermocycling format was not found to be restricted to single DNA fragment amplification. Application of the thermocycling approach to both quantitative competitive PCR (simultaneous amplification of target and competitor DNA) and cycle sequencing reactions (simultaneous amplification of dideoxy terminated fragments) was successful. This sets the stage for implementing DNA thermocycling into a variety of microfabricated formats for rapid PCR fragment identification and DNA sequencing.

DNA↗

PubMed1998

Capillary electrophoresis of insulin-like growth factors: enhanced ultraviolet detection using dynamically coated capillaries and on-line solid-phase extraction.

Insulin-like growth factor-I and -II (IGF-I and IGF-II) are difficult to separate and measure as a result of their homology, both structurally and immunologically. A number of binding proteins (BPs) which interact with the IGFs with high affinity complicate the ability to measure the IGFs accurately and reproducibly. Current methodology for measuring IGF is immuno-based and involves dissociation from the IGFs and removal of the binding proteins through sample acidification and removal by solid-phase adsorption. However, the net result is an assay that is time-consuming and, at best, semiquantitative. In an attempt to improve the reproducibility and accuracy of IGF-I and -II measurement, electrophoretic systems employing dynamically coated and bare silica capillaries were evaluated. Separations in bare silica capillaries in the presence or absence of the cationic additive, decamethonium bromide were ineffective for resolving IGF-I and IGF-II. However, when the capillary was coated dynamically with polybrene, IGF-I and -II could be resolved in a BSA sample matrix using a low pH buffer. Despite the fact that the IGFs could be resolved in the presence of an IGF-I analog used as an internal standard, polybrene recoating was required after as few as 12 runs and poor coating-to-coating reproducibility was observed. Use of polydiallyldimethylammonium chloride (PDMAC) as a dynamic cationic coating and a low pH buffer containing 0.5% PDMAC was found to be much more effective, providing reproducible separation of IGF-I and -II. It was found that PDMAC need not be included in the separation buffer to obtain reproducible analyses regarding IGF separation. Subsequently, functionality remained intact for as many as 35-40 consecutive analyses before recoating was required. Without the need for PDMAC in the buffer, on-line solid-phase extraction-capillary electrophoresis could be accomplished for detection of IGF-I and -II at concentrations as low 195 ng/ml.

Buffers↗

PubMed1998

Identification of mimotopes for the H4 minor histocompatibility antigen.

The H4 minor histocompatibility antigen (HA) of mice includes a single immunogenic peptide presented by H-2Kb molecules that stimulates skin allograft rejection and is immunodominant in the stimulation of cytolytic T lymphocytes (CTL) specific for multiple minor HA. We have identified H4 mimotopes that are recognized by the H4-specific M9 CTL clone through the use of a random peptide library comprised of bacterial clones expressing an inducible fusion protein tailed with the octamer sequence SXIXFXXL. Eight discrete mimotopes were identified that sensitized RMA-S cells for lysis by M9 CTL down to concentrations of 10(-11) M. Comparable reactivity was observed with a short-term, H4-specific CTL line indicating that the mimotopes were not solely specific for the selecting M9 clone. All mimotopes included Gly at p2 and either Val or Ile at p4, suggesting a requirement for a hydrophobic residue with specific conformation. All mimotopes included either Arg or His at p7, implicating a requirement for a specific positively charged amino acid at that position. The sixth position was more variable with four of eight mimotopes having a Val residue with single mimotopes including alternative amino acids, the majority of which were hydrophobic. Analysis of mimotopes for hydrophobicity and charge by reverse-phase HPLC and capillary electrophoresis respectively indicated that (i) mimotopes with Val at both p4 and p6 were hydrophobically similar (but not identical) to the natural H4 peptide, and (ii) a S --> E substitution at p1 resulted in a peptide (EGIVFVRL) with charge characteristics equivalent to those of the natural H4 peptide.

Amino Acid Sequence↗

PubMed1998

Direct quantitation of RNA transcripts by competitive single-tube RT-PCR and capillary electrophoresis.

Attempts are frequently made to semiquantitate mRNA as a means of circumventing the laborious and time-consuming process of quantitation that is inherent in the use of competitor templates. However, semiquantitative approaches present the risk of generating non-reproducible data due to tube-to-tube variability and/or misinterpretation of quantities of product being generated during the plateau phase of PCR. Subsequently, it is difficult to compare semiquantitative data from separate experiments, and comparisons of levels of mRNA transcript from genes that amplify with different primer pairs cannot be made. Thus, reliable methods for mRNA quantitation continue to rely on the use of internal standardization. In this report, we describe a strategy for dependable quantitation of low-abundance mRNA transcripts based on quantitative competitive reverse transcription PCR (QC-RT-PCR) coupled to capillary electrophoresis (CE) for rapid separation and detection of products. Recommendations are included for the design of RNA competitors that can be paired with target RNA for cDNA synthesis primed with a gene-specific primer; these synthesized cDNAs are then co-amplified directly in the same tube using a single primer pair. We describe (i) a protocol for a single-tube RT-PCR that provides for cDNA synthesis and subsequent PCR amplification of target and competitor in identical reaction environments at each critical enzymatic step, (ii) a unique hot-start provision for optimizing precise and consistent PCR amplifications and (iii) a method for rapid PCR product separation, detection and quantitation by CE and laser-induced fluorescence.

Animals↗

PubMed1997

Hydrophobic peptide mapping of clinically relevant heptathelical membrane proteins by capillary electrophoresis.

The structural investigation of G protein-coupled receptors has been hindered by the lack of techniques to effectively resolve the hydrophobic peptides obtained by chemical or proteolytic cleavage, as well as the minute amounts of protein typically isolated. We have developed a capillary electrophoresis method for efficient separation of hydrophobic peptides using a cyanogen bromide digest of bacteriorhodopsin as a model for these clinically important membrane proteins. This procedure includes (i) solubilization of the protein digest in acetic acid; and (ii) electrophoresis using an acetic acid-based buffer system augmented by acetonitrile and hexane sulfonic acid, in a Polybrene-coated fused silica capillary. The potential for detection sensitivity to be increased at least 100-fold by use of on-line solid-phase extraction on C18-silica is shown. This approach is potentially useful for peptide fingerprinting of sparse and extremely hydrophobic membrane receptors.

Bacteriorhodopsins↗

PubMed1997

Brain-infiltrating cytolytic T lymphocytes specific for Theiler's virus recognize H2Db molecules complexed with a viral VP2 peptide lacking a consensus anchor residue.

Mice expressing the H2b haplotype are resistant to infection with Theiler's murine encephalomyelitis virus (TMEV), which causes chronic demyelination in susceptible mice. The prominent cytolytic T-lymphocyte (CTL) response to the VP2 antigen encoded by TMEV led us to the identification of a class I-binding peptide derived from the VP2 antigen. Escherichia coli transformants overexpressing a series of 11 overlapping VP2 protein fragments were subjected to lysis and alkali digestion, and the resultant peptide pools were tested for their abilities to sensitize RMA-S targets for lysis by CTLs. The source of effector CD8+ T cells for the assays was either freshly harvested central nervous system-infiltrating lymphocytes (CNS-IL) or CNS-IL-derived VP2-specific CTL clones and lines. A 10-residue peptide at VP2 positions 121 to 130 (VP2(121-130)) (FHAGSLLVFM) was identified that sensitized targets for lysis and formed stable complexes with H2Db class I molecules. The VP2(121-130) peptide sensitized target cells for lysis by freshly harvested CNS-IL CTLs at femtomolar concentrations. Despite its relative high level of biological activity, the VP2(121-130) peptide is distinguished from other Db-binding peptides by its lack of an asparagine residue at position five, which had been previously proposed to be a requirement for Db-peptide complexing.

Amino Acid Sequence↗

PubMed1996

Mechanism of peptide separations by solid phase extraction capillary electrophoresis at low pH.

A device for on-line extraction and concentration of peptides from a dilute sample matrix prior to direct capillary electrophoretic analysis is described. The technique, termed solid phase extraction capillary electrophoresis (SPE-CE), can facilitate analysis of peptides in the low nanograms per milliliter range. Peptides from a sample matrix are adsorbed on a reversed phase resin (C-8 or C-18) cartridge in-line with an uncoated fused-silica capillary and subsequently released for free zone electrophoresis by injection of an organic elutant. Unlike previous designs and commercially available packed-inlet capillaries, the device is easily constructed from common laboratory materials and is applicable to a wide range of conventional instrumentation and methods. This device and method has been developed for use in our laboratory as a stand-alone preparative technique, specifically to provide a second-dimensional orthogonal separation of biologically derived HPLC fractions of peptides in a single analysis. To this end, extensive effort was required in both device construction and method development to attain the successful separations which are reported in this study. Extractions of dilute peptide mixtures from sample injections exceeding, but not limited to, 20 times (48 microL) the capillary volume with apparent recovery greater than 80% are shown. The selectivity of extraction of individual components of a very dilute peptide mixture (31 ng/mL with 280 microL of sample injected) is presented. The ability to efficiently extract the individual peptides from the sample was found to be concentration-dependent for the individual peptide components over a 1600-field dilution of a common calibration mixture of nine model peptides. Varying the injected volume of elution buffer demonstrated the importance of minimizing the amount of buffer used to desorb peptides to maximize the resolution of individual peptides. This study highlights implementation for direct SPE-CE for peptide analysis and discusses for SPE tip-induced mechanism through which reversal in electoosmotic flow occurs.

Amino Acid Sequence↗

PubMed1995

Concentration and separation of hypoglycemic drugs using solid-phase extraction-capillary electrophoresis.

Solid-phase extraction-capillary electrophoresis (SPE-CE) is a technique whereby very dilute analytes may be selectively extracted from a sample matrix and concentrated on-line for analysis. This study describes the first phase in the development of a method exploiting this technique for the direct analysis of hypoglycemic drugs in urine. Effective separation and detection of six sulfonylurea drug standards at concentrations below the detection limit of conventional capillary electrophoretic techniques is shown to be attainable. Since surfactant interfered with the on-line concentration process, non-MEKC (micellar electrokinetic chromatography) separation conditions were defined. Using 250 mM borate/5 mM phosphate at pH 8.4, all drugs in a mixture at 285 ng/ml were effectively extracted, concentrated from an injected volume of 2.5 microliters, non-selectively desorbed with an organic-based elution buffer and electrophoretically resolved. Sample loading was found to be linear in the 0.12-1.9 microliters range and drugs in a volume of up to 190 microliters could be concentrated and detected with a sensitivity of approximately 5 ng/ml. Not only was resolution of the desorbed material uncompromised by the presence of the SPE-tip, but separation of glipizide and glyburide was observed despite the fact that these drugs were unresolved under the same separation conditions by standard capillary zone electrophoresis (CZE). From these results, it is clear that SPE-CE not only increases the sensitivity for detection but that selectivity may be altered due to chromatographic processes occurring on the solid-phase resin.

Electrophoresis, Capillary↗

PubMed1995

Sensitivity enhancement and second-dimensional information from solid phase extraction-capillary electrophoresis of entire high-performance liquid chromatography fractions.

A novel separation technique is demonstrated for peptide analysis of entire 100 microL high-performance liquid chromatography (HPLC) fractions in a single, low pH, capillary zone electrophoresis (CZE) separation. The method employs a hybrid capillary consisting of a removable inlet section containing a reverse-phase packing and a standard CZE bare-fused silica separation capillary. The packed tip allows sample to be concentrated at flow rates greater than 20 microL/min and released in a controlled manner for CZE separation. Separations are comparable to standard CZE with minor modifications in selectivity. Using a mixture of model peptides, the hybrid capillary is shown to effect enhancement in sensitivity of > or = 100-fold. Five fractions from the HPLC separation of peptides obtained from the digestion of bovine serum albumin (BSA) were analyzed by the solid phase extraction-capillary electrophoresis (SPE-CE) method which was shown to provide rapid second-dimensional information. Practical concentration limits of detection for peptides are calculated to be 1-10 ng/mL with this technology.

Animals↗