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.

Capillary electrophoresis: a powerful microanalytical technique for biologically active molecules.

Capillary electrophoresis (CE) is a versatile microanalytical technique that has gained much attention, particularly from those working with biologically active molecules. Its appealing characteristics include unprecedented sensitivity and the ability for automating the rapid electrophoretic separation of a number of low-volume samples in a reproducible manner, with relatively short analysis times. The picomole-femtomole (10(-12)-10(-15) mol) sensitivity of UV-CE has been enhanced tremendously by the interfacing of detection systems such as laser-induced fluorescence, which has extended the sensitivity into the attomole-zeptomole (10(-18)-10(-21) mol) range. Fluorescence detection has shown great potential for the CE analysis of a wide range of biomolecules including peptides, proteins and DNA. CE research and development has taken on directions focused primarily on improving detection, understanding and exploiting the basic chemistry of CE and devising new applications.

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.

High-resolution glycoprotein analysis using capillary electrophoresis.

The high-resolution separation achievable with capillary electrophoresis has been applied successfully to the analysis of glycoproteins. Inherent in the implementation of this technology for glycoprotein analysis is the use of specific buffer additives. Bifunctional cationic reagents, such as simple alkyl chains bearing terminal amino or quaternary ammonium groups, have been particularly useful for the analysis of ovalbumin, an excellent model glycoprotein. Although dynamic coating of the capillary wall and the subsequent decrease in protein-wall interactions in known to be key in the effectiveness of these additives, much remains to be learned regarding the mechanism through which they function.

Utility of high resolution capillary electrophoresis for monitoring peptide homo- and hetero-dimer formation.

The monomer and disulfide-linked homo-dimer of two different peptides, one with an amino-terminal cysteine, the other with a cysteine at the carboxy-terminal, are shown to be baseline resolved by capillary electrophoresis in less than 15 min. Time-course for homo-dimer formation with both peptides, either under mild (air) or stronger (hydrogen peroxide oxidizing conditions, was easily monitored. Confirmation that the second peak appearing under oxidizing conditions was indeed the homo-dimer was obtained with mass spectrometry. The possibility that stronger oxidizing conditions led to the production of the sulfonic acid derivative of the monomeric peptide, was ruled out through generation of the derivative by performic acid oxidation. As expected, the negative charge of the sulfonic acid moiety gives the peptide a slower electrophoretic mobility than both the monomer and the dimer. Moreover, as would be expected with a sulfonic acid derivative, oxidation to the dimeric form was not possible. This was consistent with the observation that the homo-dimer peak could be reduced to monomeric form in the presence of dithiothreitol. Co-oxidization of the amino- and carboxy-terminal peptides led to the expected production of both homo-dimers and the hetero-dimer, all of which were resolved.

Capillary electrophoresis of DNA potential utility for clinical diagnoses.

The last few years have witnessed a tremendous shift in the use of capillary electrophoresis for clinical applications, particularly with DNA analysis. As a result of the large number of DNA-based clinical assays, there is an intense interest in making DNA analysis faster, less expensive and more automated. We describe the evaluation of CE-based single-strand conformation polymorphism (SSCP) and dideoxy fingerprinting (ddF) analysis for the detection of single-point mutations within a Mycobacterium tuberculosis-specific amplified DNA fragment. Both were found to be capable of detecting the mutation in the resistant isolate but ddF showed the most promise with respect to specificity and ease of implementation. In addition, initial results with a CE-based sizing method is shown to be competitive and, perhaps, superior to a Southern blot analysis for the detection of hepatitis C viral (HCV) infection.

Multiple-buffer-additive strategies for enhanced capillary electrophoretic separation of peptides.

A dodeca-peptide from the beta-subunit of thyroid stimulating hormone (TSH) was used as a model system for developing "multiple-buffer-additive" strategies to effect the separation of structurally-similar peptides. A series of synthetic peptides included six peptides with identical amino acid composition and two with multiple alanine substitutions at selected positions. Those with identical amino acid composition included the native and reverse sequences of residues 101-112 of the beta-TSH and four "computer-shuffled" amino acid sequences. Buffer additives such as acetonitrile (ACN), hexane sulfonic acid (HSA), and hexamethonium bromide (HxMBr), were shown to alter selectivity dramatically. HSA, an ion-pairing agent, and ACN, known to alter the hydrophobic environment of the solute, and HxMBr, which is thought to negate solute-wall interactions, are shown to independently effect only partial resolution of the mixture. It is shown that only with the proper combination of HSA and ACN are all mixture components resolved. These results re-affirm that CE selectivity may be altered by changes in buffer ionic strength or with the addition of HSA, but also show that further changes in selectivity can be achieved through alteration of buffer hydrophobicity. The observed changes in selectivity accompanying the addition of HSA and ACN may be due to differing electrophoretic mobilities resulting from nearest-neighbor effects or subtle differences in peptide secondary structure or solvation. This emphasizes the importance of employing multiple-buffer-additive strategies for effecting the resolution of peptide mixtures that are difficult to separate.

Rapid capillary electrophoretic analysis of human serum proteins: qualitative comparison with high-throughput agarose gel electrophoresis.

This study details the qualitative results from a comparative evaluation of agarose gel electrophoresis (AGE) and (CZE) as a screening procedure for monoclonal proteins in serum. Three hundred and five serum samples were analyzed by the two techniques; samples suspected of containing monoclonal proteins based on abnormalities observed with AGE or CZE were confirmed by immunoelectrophoresis and/or immunofixation. CZE separation conditions were simple (requiring only a bare silica capillary and 150 mM borate buffer, pH 10.0) and separation was complete in under 120 s. The results obtained by CZE were comparable or better than those obtained with AGE. Samples displaying "point-of-application" artifacts on AGE were not problematic by CZE analysis; an abnormal profile, due to the presence of a monoclonal protein, or a normal profile were clearly observable. The rapid analysis, excellent reproducibility, automation and relatively high throughput (approximately 90 samples per 8 h on a single instrument) sets the stage for CZE analysis of serum proteins to be used routinely in a clinical setting.

Capillary electrophoresis in biotechnology.

Capillary electrophoresis (CE) is a versatile micro/macroanalytical technique gaining widespread usage for the separation and analysis of ionic substances. It has captured the attention of those working in a variety of arenas focused on biologically-active molecules. Its appealing characteristics include unprecedented mass sensitivity and the ability for precise, automated electrophoretic separation of microvolume samples with relatively short analysis times. Such versatility in bioanalysis makes it an inviting replacement for some of the labor-intensive, time-consuming methodologies performed via electrophoretic gels. Moreover, CE compliments the ease and speed of HPLC while eliminating the problem of excessive solvent volume usage and hazardous waste disposal. Further attractive characteristics of this technology include the analyses of a diverse spectrum of analytes, ranging from small organic ions to macromolecular protein complexes and DNA. While combining some of the most robust aspects of traditional electrophoresis, chromatography, and capillary technology, recent CE research and development has focused on avenues leading to improving detection and understanding and employing the basic chemistry of CE vis-a-vis new applications.

Select cardiovascular and metabolic responses of diabetic rats to moderate exercise training.

The combined influence of diabetes and moderate treadmill exercise training on select metabolic and cardiovascular parameters was investigated with mature male Sprague-Dawley rats assigned to either control diabetic or diabetic groups receiving exogenous insulin. Experimental diabetes was induced with streptozotocin (80 mg.kg-1, i.v.) and verified by blood glucose concentrations greater than 16 mmol. The animals were designated as control, insulin-injected (5 U.kg-1, twice daily), or saline-injected (twice daily), and assigned to either non-trained or trained sub-groups. Insulin treatment partially restored the measured physiological functions to within normal limits. All animals were trained at 60 to 70% maximal oxygen consumption for 9 wk and exhibited higher maximal oxygen consumption values and cytochrome oxidase activity of the soleus muscles. Diabetes caused lower (P less than 0.05) reductions in resting heart rate but training-induced bradycardia did not occur in any group. Heart rate response to atropine sulfate (1 mg.kg-1, atrial choline acetyltransferase activity, atrial acetylcholine concentration, and quinuclidinyl benzilate binding was measured to evaluate changes in the parasympathetic nervous system. Atropine-induced cardiac acceleration was most pronounced in control and least effective in diabetic animals. Endurance training had no meaningful influence on this response to cholinergic inhibition. Quinuclidinyl benzilate binding for the diabetic and the diabetic groups receiving insulin revealed no change in receptor number, receptor affinity, or training effects. These findings indicated that 9 wk of exercise training improves the aerobic capability of insulin-deficient rats without changing cardiovascular characteristics associated with the parasympathetic nervous system.

A microcomputer program for determining turnover rates before and after intervention in a single animal.

A set of three programs to calculate the turnover of biomolecules whose metabolism follows a steady-state precursor-product relationship and follows the open, single-compartment kinetics model has been written in MS-BASIC. The programs comprise a system for determining two values of turnover, before and after an intervention which may alter the turnover rate, in a single animal. The programs have been extensively tested in our laboratory for the determination of norepinephrine turnover under differing physiological and pharmacological conditions. The utility of the programs lies in their ability to readily adapt to turnover determinations for any substance whose metabolic pathway conforms to the model constraints. This includes the biogenic amine neurotransmitters, peptides and proteins, and many small biological molecules or pharmacological agents.

Effect of cationic buffer additives on the capillary electrophoretic separation of serum transferrin from different species.

The presence of specific transferrin (Tf) glycoforms in human serum has been shown to correlate with certain clinical syndromes. Hence, the ability to separate and quantitatively measure the various forms of human transferrin has become increasingly important. As a means of evaluating the potential for developing a rapid capillary electrophoresis-based assay for the analysis of carbohydrate-deficient transferrins (CDTs), the capillary zone electrophoretic (CZE) analysis of Tfs from several species was evaluated using uncoated capillaries and a separation augmented with cationic additives. With bovine Tf, five peaks (representing different sialylated forms) were partially resolved in borate and baseline-resolved when 1,4-diaminobutane was added to the buffer. These same conditions were found to be inadequate for the resolution of the sialoforms from other species. Some success was achieved using alpha, omega-bis-quaternary ammonium alkanes instead of the 1,4-diaminobutane and optimizing the pH for each of the species Tfs. Human Tf was found to be resolved in an uncoated capillary equilibrated with a borate buffer containing millimolar concentrations of decamethonium bromide as a buffer additive. Under these conditions, resolution of the various sialoforms from the iron-saturated Tf was possible and the glycoforms were found to migrate differently than their iron-depleted counterparts. Despite the resolution achievable under these conditions, the lengthy analysis time is incompatible with the requirements for a clinical CZE-based assay.

Preliminary investigations of preconcentration-capillary electrophoresis-mass spectrometry.

Analyte preconcentration on-line with capillary electrophoresis-mass spectrometry (PC-CE-MS) is described. Preconcentration cartridges were fabricated from PTFE tubing filled with ca. 1-2 mm bed of reversed-phase C18 HPLC packing or polymeric reversed-phase beads. The particle size of the stationary phase was of larger dimension than the internal diameter of the CE capillary. Therefore, PC-CE capillaries were assembled without frit material and held together by friction. The wide applicability of on-line PC-CE-MS is demonstrated by the analysis of solutions containing peptides, proteins, and synthetic analogues of putative metabolites of the neuroleptic agent haloperidol.

Vagus nerve stimulation alters regional acetylcholine turnover in rat heart.

The turnover of neurotransmitter is a direct measure of neuronal function, varying with the impulse activity of the nerve. It is not known if vagal stimulation increases acetylcholine release uniformly throughout the heart, or if modification of neural signals occurs between the vagal nerve trunks and postganglionic synaptic terminals. The rate constant of acetylcholine turnover was measured in conduction and contractile regions of heart by quantifying the incorporation of [3H]choline into acetylcholine after labeling of the blood choline pool in urethane-anesthetized rats during two levels of vagal activity. Choline and acetylcholine were assayed by high pressure liquid chromatography with electrochemical detection of post-column enzymic reaction product, peroxide. The specific activities of choline and acetylcholine in the tissues at sacrifice were used to calculate the fractional turnover rates in cardiac regions. Supramaximal bilateral vagal stimulation for 20 minutes decreased heart rate (P less than 0.05), while mean arterial blood pressure remained constant. The rate constants for acetylcholine turnover in right atrial regions containing the sinoatrial node, left atrial tissues, and interatrial septum doubled from control values during vagal stimulation. In contrast, the fractional rate constants of acetylcholine turnover did not change in the right and left ventricles during vagal stimulation. We interpret these results to indicate general activation of postganglionic parasympathetic fibers to the atria and selective modulation of postganglionic parasympathetic neural function to the ventricles.

Presynaptic regulation of cardiac sympathetic function in guinea pigs.

Cold stress (4 degrees C) induces a pressor response and variable increases in an index of sympathetic neural function, the rate constant of norepinephrine turnover, kNE. In heart, presynaptic cholinergic muscarinic and alpha-2 adrenergic influences may contribute to regional variation in responses of kNE to cold stress. Animals were pretreated with vehicle, a muscarinic cholinergic antagonist, quinuclidinyl benzilate (QNB), an alpha-2 adrenergic antagonist, yohimbine (YOH) or combined QNB + YOH. An increase in kNE was determined from incorporation of radiolabeled tyrosine into norepinephrine in a control period at 24 degrees C and again at 4 degrees C. The increment in kNE factored by the increment in blood pressure indicated the extent of increased sympathetic function in each cardiac region. In sino-atrial node, sympathetic function was increased significantly (P less than .05) by QNB + YOH compared to other treatments, suggesting that both cholinergic and alpha-2 adrenergic presynaptic influences were important. In contrast, in right and left ventricles, YOH or QNB + YOH, but not QNB alone, increased sympathetic function significantly, suggesting that only alpha-2 adrenergic influences were important. These data support the concept that presynaptic regulation of cardiac sympathetic function differs in sino-atrial node and ventricles of guinea pigs during activation with cold stress.

Capillary electrophoresis as a clinical tool for the analysis of protein in serum and other body fluids.

Most electrophoretic analyses of proteins in the clinical laboratory are currently carried out by electrophoresis in acrylamide or agarose gels. This labor-intensive method is now being challenged by capillary electrophoresis (CE) because of its potential for automated, rapid, high efficiency separations. The automatability of CE itself, combined with its amenability to interfacing with other robotized functions, positions this technology perfectly for the analysis of proteins in physiological matrices, such as serum, urine, and cerebrospinal fluid. The focus of this overview is to familiarize the clinical scientist with the research demonstrating the applicability of the technology to the clinical protein laboratory.

Capillary electrophoretic detection of metabolites in the urine of patients receiving hypoglycemic drug therapy.

Micellar electrokinetic chromatography (MEKC) in tandem with diode array detection (DAD) has been exploited as an analytical method for the separation and detection of sulfonylurea drugs. The ultimate goal is the development of an assay to detect these drugs or their metabolites in urine as a means of diagnosing sulfonylurea drug abuse. Using a separation buffer consisting of 5 mM borate/5 mM phosphate/75 mM sodium cholate, separation of both the second and third generation sulfonylurea drugs can be achieved. The characteristic absorbance spectra associated with each of the third generation drugs, glipizide and glyburide, allow for their identification in mixtures. Coinjection of glyburide, its primary metabolite, hydroxy glyburide, and glipizide demonstrated that the metabolite was resolved from the parent drug but shared its absorbance spectral properties. MEKC analysis of a series of solid phase-extracted urine samples from patients prescribed glipizide or glyburide, as well as from control patients not ingesting the drug, showed that the parent compounds were difficult to detect in the urine. However, the use of DAD allowed for detection of metabolites in the urine of these patients. With glyburide patients, only primary metabolites were detected, while urine from patients on glipizide showed a series of peaks whose absorbance spectra was consistent with the presence of both primary and secondary metabolites. In addition, the intensity of the metabolite peaks corresponded reasonably well with the respective dose and in vivo time interval associated with the urine collection. This study shows that MEKC with DAD has potential for further exploration as a clinical assay for detecting surreptitious abuse of sulfonylurea drugs.

Determination of nitrite and nitrate reduction by capillary ion electrophoresis.

Production of nitrates and nitrites is a common step in many methodologies used to measure nitric oxide (NO) and NO-derived products in biological fluids. We report conditions that allow the rapid separation and quantification of nitrite from nitrate ions in biological fluids by capillary ion electrophoresis (CIE). CIE can be used to directly quantify nitrites and nitrates near the millimolar range. To detect lower levels, we have used CIE to monitor the reduction of nitrites and nitrates to NO for chemiluminescence detection. For reduction reactions, we directly compared the ability of three commonly used agents--potassium iodide (KI), mercuric chloride (HgCl2) and vanadium chloride (VCl3)--to reduce nitrite and nitrate ions to NO. Nitrites/nitrates can be efficiently reduced to NO at 37 degrees C using vanadium chloride (100%) or HgCl2 (80%). However, these CE-derived conditions cannot simply be extrapolated to chemiluminescence measurements. Vanadium (III) yields high background in the photomultiplier that diminishes the sensitivity of chemiluminescence measurement to that outside of physiological ranges. We find that reactions carried out at 37 degrees C in 2 M HCl using HgCl2 is efficient using both techniques.

Effective and reproducible capillary electrophoretic separation of thiols under conditions where exceptionally high current is generated.

There is an escalating interest in the role of endogenous nitric oxide (NO) in biological systems and how this chemical regulates physiology in normal and disease states. In biological systems, the cellular concentration can be estimated, in the simplest form, by accounting for NO and its common metabolites, nitrate and nitrite. However, since NO is also known to interact with other chemical entities, such as thiols, it would be valuable to have a rapid qualitative assay that could account for thiol binding and S-N bond cleavage in the presence of different reducing agents. A separation buffer consisting of 10 mM phosphate, 10 mM HCl, and 250 mM KCl is shown to be adequate for the separation of glutathione, nitrosylated glutathione, and glutathione disulfide solubilized in 2 M HCl. The current observed under these separation conditions (249 microA at 11 kV) is extremely high by capillary electrophoresis (CE) standards, with a total power (current x voltage/capillary length) calculated to be in excess of 7 W/m. While this exceeds the approximately 1.0 W/m recommended by previous studies as a maximum for CE-based separations, we demonstrate that effective CE separation of thiols can, in fact, be accomplished under these conditions with acceptable reproducibility, provided that buffer depletion issues are addressed.

Choline and acetylcholine concentration in transplanted rat heart.

A liquid chromatographic assay was used to determine the choline and acetylcholine concentrations in the four chambers of rat hearts 2, 4, and 8 days after transplantation into an abdominal site. Corresponding measurements were made in the hearts of host rats. We found regional cardiac acetylcholine concentrations in controls follow the nonuniform pattern seen with choline acetyltransferase (CAT) activity, being highest in the atria (8-12 nmol/g) and lower in the ventricles (0.7-1.6 nmol/g). Following transplantation, acetylcholine levels decreased significantly only in the right ventricle after 8 days. Following a unilateral vagotomy (random, left or right), acetylcholine concentrations in the distal portion of the severed nerve decreased to half the value of the intact contralateral side by 4 days. The continued presence of acetylcholine, despite significantly reduced CAT activity in the severed nerve and transplanted heart, suggests that acetylcholine is preserved, perhaps by vesiculation in nonstimulated postganglionic terminals. The localized decrease in acetylcholine in the right ventricle after 8 days suggests that transplantation may interrupt the postganglionic fibers to this area.