Biomedical subjects

K L Johnson

Publications and source records attributed to K L Johnson.

At least 19 recordsLinked to original sources

PubMed2001

The role of mass spectrometry in vaccine development.

For the most part, vaccine development to date has been empiric. While sometimes successful, such a strategy is 'hit or miss', and fails to advance the basic science of vaccine development. Preferable would be tools that allow for a more directed development of vaccines at either the population or sub-population level. Characteristics of useful tools in vaccine development should include the ability to identify and characterize the spectrum of antigenic peptides presented by MHC molecules to which the immune system responds by the development of protective immune responses. In addition, because the explosion in human genomics allows the ability to understand MHC haplotypes at the population level, as well as an enhanced understanding of MHC binding motifs, new tools might further allow for an understanding of which vaccine antigens are capable of being bound and presented to the immune system by MHC molecules. New mass spectrometry technology fulfils these criteria, and may well lead to a revolution in the design of new vaccines. This paper will review the basics of mass spectrometry techniques as applied to the identification and characterization of vaccine peptide antigens, and discusses how these tools can be applied to vaccine development.

Chromatography, High Pressure Liquid↗

PubMed2001

Determination of in vivo adducts of disulfiram with mitochondrial aldehyde dehydrogenase.

Extensive use for disulfiram (DSF) has been found in the aversion therapy treatment of recovering alcoholics. Although it is known to irreversibly inhibit hepatic aldehyde dehydrogenase (ALDH), the specific mechanism of in vivo inhibition of the enzyme by the drug has not been determined yet. We have demonstrated in this report a novel, but simple and rapid method for structurally characterizing in vivo derived protein-drug adducts by linking on-line sample processing to HPLC-electrospray ionization mass spectrometry (HPLC-MS) and HPLC-tandem mass spectrometry (HPLC-MS/MS). Employing this approach, rats were administered DSF, and their liver mitochondria were isolated and solubilized. Both native and in vivo DSF-treated mitochondrial ALDH (mALDH) were purified in one step with an affinity cartridge. The in vivo DSF-treated mALDH showed 77% inhibition in enzyme activity as compared with that of the control. Subsequently, the control and DSF-inhibited mALDH were both subjected to HPLC-MS analyses. We were able to detect two adducts on DSF-inhibited mALDH, as indicated by the mass increases of approximately 71 and approximately 100 Da. To unequivocally determine the site and structure of these adducts, on-line pepsin digestion-HPLC-MS and HPLC-MS/MS were performed. We observed two new peptides at MH(+) = 973.7 and MH(+) = 1001.8 in the pepsin digestion of DSF-inhibited enzyme. These two peptides were subsequently subjected to HPLC-MS/MS for sequence determination. Both peptides possessed the sequence FNQGQC(301)C(302)C(303), derived from the enzyme active site region, and were modified at Cys(302) by N-ethylcarbamoyl (+71 Da) and N-diethylcarbamoyl (+99 Da) adducts. These findings indicated that N-dealkylation may be an important step in DSF metabolism, and that the inhibition of ALDH occurred by carbamoylation caused by one of the DSF metabolites, most likely S-methyl-N,N-diethylthiocarbamoyl sulfoxide (MeDTC-SO). Finally, there was no evidence of the presence of an intramolecule disulfide bridge modification on the peptide FNQGQCCC.

Alcohol Deterrents↗

PubMed2001

Effect of enzyme inhibitors on protein quaternary structure determined by on-line size exclusion chromatography-microelectrospray ionization mass spectrometry.

Aldehyde dehydrogenases (ALDH) are a family of enzymes primarily involved in the oxidation of various aldehydes. Most ALDH enzymes derived from mammalian sources have been shown to exist as homotetramers, consisting of four identical subunits of approximately 54 kDa. The presence of the homotetramer appears to be necessary for enzyme activity. In this study, recombinant rat liver mitochondrial ALDH (rmALDH) was inhibited in vitro with four different inhibitors, namely, disulfiram (MW, 296.5), prunetin (MW, 284.3), benomyl (MW, 290.3), and N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) (MW, 351.8). Subsequently, inhibited rmALDH was analyzed by a novel approach of on-line size exclusion chromatography-microelectrospray ionization-mass spectrometry (SEC-muESI-MS) to examine the noncovalent quaternary structural stability of the inhibited enzyme. Analysis of native rmALDH by SEC-muESI-MS revealed predominantly the homotetramer (Mr = approximately 217,457 Da, +/- 0.01%) with some in-source, skimmer-induced dissociation to afford monomer (Mr = approximately 54,360 Da, +/- 0.01%). Both disulfiram and prunetin inhibited rmALDH by >70% and >90%, respectively, but did not disrupt the quaternary structure of rmALDH. Furthermore, there was no detectable change within experimental error (+/- 0.01%) of the disulfiram or the prunetin homotetramers (Mr = approximately 217,448 Da and Mr = approximately 217,446 Da). This may possibly indicate that inhibition occurred via formation of intramolecular disulfide bond at the enzyme active site, or weak affinity noncovalent binding. In contrast, benomyl-inhibited rmALDH homotetramer (>90% inhibition) exhibited a Mr = approximately 217,650 Da (+/- 0.01%) corresponding to two butylcarbamoyl adducts on two of the four enzyme subunits. The skimmer-induced monomer afforded a mixture of unmodified rmALDH (Mr = approximately 54,365 Da, +/- 0.01%) and butylcarbamoylated enzyme (Mr = approximately 54,459 Da, +/- 0.01%). Finally, TPCK (>90% inhibition) modified all four subunits of rmALDH to give Mr = approximately 218,646 Da (+/- 0.01%). In all four cases while significant enzyme inhibition occurred, no destabilization of the quaternary complex was detected.

Aldehyde Dehydrogenase↗

PubMed2000

Isolation and rapid identification of an abundant self-peptide from class II HLA-DRB1*0401 alleles induced by measles vaccine virus infection.

Class II HLA-DR genes play an important role in the immune response to viral antigens. The effect of measles vaccine virus (MVV) infection on the induction of self-peptides presented by HLA-DR molecules during the immune response to viral infection is poorly known. Here, we describe a strategy for isolation and rapid sequence determination of an MVV-inducible class II bound peptide from a membrane protein (Leu-13). Peptides bound to HLA-DR4 (DRB1*0401 peptide complex) were eluted from immunoaffinity-purified HLA-DR4, peptides were differentially screened by MALDI-TOF-MS and subsequently sequenced by post source decay (PSD)-MALDI-TOF-MS. Human B-cells infected with MVV demonstrated an enhanced pattern of self-peptide production after MVV infection. This relatively simple analytical protocol provides a sensitive method for the direct identification of peptides associated with MHC class II DR molecules. More broadly, this same approach can be used to identify sequences of specific MVV processed peptides presented by any class II MHC DR molecule.

Alleles↗

PubMed2000

Identification of the protein-drug adduct formed between aldehyde dehydrogenase and S-methyl-N,N-diethylthiocarbamoyl sulfoxide by on-line proteolytic digestion high performance liquid chromatography electrospray ionization mass spectrometry.

Disulfiram has been used clinically as an aversion therapy treatment for recovering alcoholics. One of its metabolites, S-methyl-N, N-diethylthiocarbamoyl sulfoxide (MeDTC-SO), is currently believed by some to be the active metabolite in vivo. We demonstrate in this report that MeDTC-SO is a potent irreversible inhibitor of recombinant rat liver mitochondrial aldehyde dehydrogenase (rlmALDH), the enzyme responsible for oxidizing acetaldehyde formed during ethanol metabolism. Recombinant rlmALDH was inhibited by MeDTC-SO after in vitro incubation with an IC(50) = 4.62 microM. The inhibition of rlmALDH was found to be accompanied by a concomitant increase of approximately 100 Da to the molecular mass of the native enzyme as determined by on-line high performance liquid chromatography (HPLC) electrospray ionization mass spectrometry (LC/MS), indicating that a covalent modification has occurred. To determine the site and structure of this covalent adduct, we developed a novel approach to characterize specific protein-drug interactions by linking a proteolytic enzyme digestion cartridge on-line with LC/MS. The on-line pepsin digestion LC/MS of MeDTC-SO-inhibited rlmALDH revealed an ion at MH(2)(2+) = 500.9, which was not present in the pepsin digestion of the non-inhibited enzyme. This peptide was tentatively attributed to the putative active site peptide (FNQGQC(301)C(302)C(303)) plus the adduct. This peptide was subjected to analysis by LC/MS/MS, which allowed us to determine that the covalent modification was associated with a single carbamoyl adduct at Cys-302, which has been shown to be the active site nucleophile of the enzyme.

Alcohol Deterrents↗

PubMed2000

Female fetal cells in maternal blood: use of DNA polymorphisms to prove origin.

The nucleated erythrocyte (NRBC) is one of the target fetal cell types for noninvasive genetic diagnosis using maternal peripheral blood. However, it is now known that pregnancy can stimulate the production of maternal NRBCs. When isolating female gamma-positive NRBCs, fluorescence in situ hybridization (FISH) analysis may show two X chromosome signals per nucleus, and therefore it cannot be conclusively determined whether the isolated cells are fetal or maternal in origin. The purpose of this study was to develop a means of verifying that a female cell is fetal on the basis of polymorphic short tandem repeat markers. Peripheral blood samples were obtained from women who had just undergone termination of pregnancy. Nucleated candidate fetal cells were isolated by flow-sorting using antibody to the gamma-chain of fetal hemoglobin and Hoechst 33342. FISH analysis was performed using X and Y chromosome specific probes. Female gamma-positive cells and leukocytes were micromanipulated separately and subjected to fluorescent polymerase chain reaction amplification of chromosome 21 and/or 18 STR markers (D21S11, D21S1411, D21S1412, and D18S535). In all ten cases analyzed, the gamma-positive female candidate fetal cells were determined to be fetal in origin by the presence of shared and nonshared DNA polymorphisms when compared with maternal leukocytes. These results show that genetic analysis can be performed on all fetal NRBCs, including female fetal cells that cannot be distinguished from maternal cells based on FISH analysis alone.

Cell Separation↗

PubMed2000

Nitric oxide is a mediator of the late-phase response in an animal model of nasal allergy.

The presence of nitric oxide (NO) in the nose is well documented; however, the role of this molecule in nasal physiology is still poorly understood. Our laboratory has previously demonstrated that NO is a mediator of the immediate secretory response to an intranasal histamine challenge in a rat model of nasal allergy. Histamine challenge, however, does not elicit a late-phase response (LPR). To study the role of NO in the LPR, we developed a model of nasal allergy in which brown Norway rats are actively sensitized to the allergen ovalbumin and later challenged intranasally with either phosphate-buffered saline solution (vehicle), ovalbumin in vehicle, or ovalbumin and the NO synthase inhibitor N -nitro-l -arginine methyl ester. In each experiment, nasal lavage samples were collected 30, 120, 240, and 360 minutes after challenge. Lavage samples were analyzed for albumin content by ELISA, inflammatory cell concentration with a hemocytometer, and evidence of inflammation by light microscopy. Blocking NO synthesis with N -nitro-l -arginine methyl ester significantly inhibited both albumin exudation and inflammatory cell influx into the nasal cavity during the LPR. These data suggest that NO plays a role in the LPR of nasal allergy.

Albumins↗

PubMed2000

Effect of stenting after laryngotracheal reconstruction in a subglottic stenosis model.

OBJECTIVES: This study addresses the impact of stenting on early wound healing after laryngotracheal reconstruction (LTR) in a rabbit model with established subglottic stenosis. METHODS: Subglottic stenosis was created in 42 New Zealand white rabbits through a transoral, endoscopic technique. Three weeks later, endoscopy and axial CT were performed to document and grade the degree of stenosis. Subsequently, LTR was performed in all animals, with half of the animals receiving an intraluminal stent. Four rabbits from each group were euthanized on postoperative days 6, 9, 14, 21, and 28. Measurements of graft vascularization were obtained with a computerized image measurement program, and a comparison was made regarding the rate of vascularization. RESULTS: There was a statistically significant increase in the rate of vascularization in the stented group (mean 75% +/- 5% vs 56% +/- 3% at day 14; P < 0. 05). However, clinical and radiographic comparisons of the stented and nonstented specimens revealed a trend toward increased mucosal edema and granulation tissue in the stented group at later time intervals (days 21 and 28). CONCLUSION: This analysis suggests that stenting does not inhibit graft vascularization in the early postoperative period after LTR; however, complications were seen in the stented group at longer time intervals.

Animals↗

PubMed2000

The classical arabinogalactan protein gene family of arabidopsis.

Arabinogalactan proteins (AGPs) are extracellular proteoglycans implicated in plant growth and development. We searched for classical AGPs in Arabidopsis by identifying expressed sequence tags based on the conserved domain structure of the predicted protein backbone. To confirm that these genes encoded bona fide AGPs, we purified native AGPs and then deglycosylated and deblocked them for N-terminal protein sequencing. In total, we identified 15 genes encoding the protein backbones of classical AGPs, including genes for AG peptides-AGPs with very short backbones (10 to 13 amino acid residues). Seven of the AGPs were verified as AGPs by protein sequencing. A gene encoding a putative cell adhesion molecule with AGP-like domains was also identified. This work provides a firm foundation for beginning functional analysis by using a genetic approach.

Amino Acid Sequence↗

PubMed2000

Temporal integration in the presence of off-frequency maskers.

Temporal integration was measured at a relatively low and a relatively high signal frequency under conditions of off-frequency masking. The masker was typically gated for 300 ms, and the signal was presented 70 ms after masker onset. In experiment 1, the signal frequency was 500 or 2000 Hz. Temporal integration was measured in quiet and in the presence of a masker whose frequency was lower or higher than the signal frequency. In all listening situations, there was less integration at 2000 Hz than at 500 Hz. This effect of frequency was particularly dramatic in the presence of a lower frequency masker, where there was almost no integration at 2000 Hz. Experiment 2 showed that this dramatic effect of frequency cannot be understood in terms of the underlying psychometric functions. Experiment 3 measured temporal integration at 750 and 2000 Hz for a large number of masker-signal frequency separations for both a tonal and a noise masker, and in conditions where the masker was gated or continuous. The results with the gated tonal masker largely confirmed the results of experiment 1. The results with the continuous tonal masker and the gated or continuous noise masker, however, were quite different. In those cases, the amount of temporal integration at both signal frequencies was more or less independent of the masker-signal separation; the masked temporal integration was nearly equal to the integration in quiet. Thus based on the conditions evaluated here, off-frequency masked temporal integration differs substantially from integration in quiet only for gated tonal maskers located considerably lower in frequency than the signal. It is unclear how to account for this finding, although it may be related to attentional factors.

Adult↗

PubMed2000

The use of fluorescence in situ hybridization (FISH) on paraffin-embedded tissue sections for the study of microchimerism.

We describe here a simple and versatile method of fluorescence in situ hybridization (FISH) on paraffin-embedded tissue sections with specific application in the study of microchimerism, that is, the presence of intact foreign cells within an individual. This is accomplished through the use of X and Y chromosome-specific probes to identify the presence of male nuclei within a tissue section from a female, and vice versa. This technique requires only minor modification if at first the hybridization does not yield fluorescent signals of high quality. Analysis of a wide variety of tissue types is possible with this method, and multiple tissue types from one or more individuals can be processed in the same hybridization reaction. This robust FISH method has been used successfully in our laboratory to investigate fetal cell microchimerism in the following paraffin-embedded tissue types: skin, lung, thyroid, adrenal gland, lymph node, heart, spleen, liver, pancreas, kidney, and intestine.

Cell Nucleus↗

PubMed2000

A review of fluorescence in situ hybridization (FISH): current status and future prospects.

Fluorescence in situ hybridization (FISH) is a powerful technique for detecting DNA or RNA sequences in cells, tissues and tumors. This molecular cytogenetic technique enables the localization of specific DNA sequences within interphase chromatin and metaphase chromosomes and the identification of both structural and numerical chromosome changes. FISH is quickly becoming one of the most extensively used cytochemical staining techniques owing to its sensitivity and versatility, and with the improvement of current technology and cost effectiveness, its use will surely continue to expand. Here we review the wide variety of current applications and future prospects of FISH technology.

Animals↗

PubMed2000

Differential effects of interleukin-3 on fetal and adult erythroid cells in culture: implications for the isolation of fetal cells from maternal blood.

Fetal clonogenic erythroid cells may be present in maternal blood and serve as a source of fetal DNA for prenatal genetic diagnosis. Proliferating nucleated red cells in cultures from first and second trimester fetal blood contain only fetal haemoglobin (HbF; F+A- cells), whereas nucleated red cells from adult blood contain also adult haemoglobin (HbA; F+A+ or F-A+ cells). Thus, fetal red cells can be identified and flow sorted. However, a few adult cells are also F+A-, which reduces the purity of fetal cell isolation. Culture media optimized for erythropoiesis contain interleukin-3 (IL3). We show here that IL3 strongly stimulates the growth of F+ cells (both F+A- and F+A+) in cultures from adult blood but has only a comparatively small effect on F+A- cells in cultures from fetal blood. This difference is maintained in the unified conditions of co-cultures of adult and fetal cells, so that the purity of fetal cell sorts can be increased by omitting IL3 from the culture medium. We further show that IL3 accelerates the exhaustion of the long-term division potential of adult cells, allowing fetal secondary colonies to be identified by their size following a two-stage culture scheme. Thus, the choice to omit or include IL3 in the growth medium of maternal blood cultures should depend on whether fetal nucleated red cells are to be isolated by flow sorting after one week, or by picking secondary colonies after a later secondary culture stage.

Adult↗

PubMed1999

The distribution of chromosome damage, non-reciprocal translocations and clonal aberrations in lymphocytes from Chernobyl clean-up workers.

In this paper we determined whether the frequencies of translocations and insertions are proportional to chromosome size in peripheral blood lymphocytes from Chernobyl nuclear accident clean-up workers and healthy unexposed control subjects. The frequency of aberrations among chromosomes 1, 2 and 4 in both groups was found to be significantly different from the distribution expected on the basis of chromosome size, although the difference was only marginally significant in controls. We also determined whether differences exist in aberration frequencies measured by two scoring systems: the classical method, where reciprocal exchanges are scored as one event, and PAINT, where each break junction is scored as a single event. The two scoring systems gave highly correlated results which yielded an interpretable arithmetic relationship between frequency measurements using the two systems. Approximately 34% of all translocations were observed to be non-reciprocal, and cells bearing clones of abnormal cells were observed in 6 of 198 subjects (3.0%). Our results demonstrate that clones of abnormal cells and the presence of non-reciprocal translocations contribute to the non-proportional distribution of radiation-induced and spontaneous cytogenetic damage.

Chromosome Breakage↗

PubMed1999

Unspecific activation of caspases during the induction of apoptosis by didemnin B in human cell lines.

Caspases have been implicated in the induction of apoptosis in most systems studied. The importance of caspases for apoptosis was further investigated using the system of didemnin B-induced apoptosis. We found that benzyloxycarbonyl-VAD-fluoromethylketone, a general caspase inhibitor, inhibits didemnin B-induced apoptosis in HL-60 and Daudi cells. Acetyl-YVAD-chloromethylketone, a caspase-1-like activity inhibitor, inhibits didemnin B-induced apoptosis in Daudi cells, whereas the caspase-3-like activity inhibitor, acetyl-DEVD-aldehyde, has no effect. Using immunoblots to investigate cleavage of caspases-1 and -3, we found that both caspases are activated in both cell lines. We showed that the caspase substrate poly(ADP-ribose)polymerase is cleaved in these cells after didemnin B treatment. In both cell lines, poly(ADP-ribose)polymerase cleavage is inhibited by benzyloxycarbonyl-VAD-fluoromethylketone and also by acetyl-YVAD-chloromethylketone in Daudi cells. These results indicate that a caspase(s) other than caspase-3 is required for didemnin B-induced apoptosis. We show that caspases may be activated during apoptosis that are not required for the progression of apoptosis.

Amino Acid Chloromethyl Ketones↗

PubMed1999

Analysis of lipophilic peptides and therapeutic drugs: on-line-nonaqueous capillary electrophoresis-mass spectrometry.

This minireview addresses the usefulness of nonaqueous capillary electrophoresis-mass spectrometry (NACE-MS), mainly in the analysis of lipophilic peptides such as gramicidin S and bacitracin, and therapeutic drugs such as pyrazoloacridine, the H2-antagonist mifentidine, tamoxifen, and their metabolites. The beneficial effects of NACE-MS in typical bioanalytical applications are analyzed case by case. A suitable and widely applicable NACE-MS analysis is identified, which is an electrolyte buffer containing ammonium acetate (5-50 mM) and/or acetic acid (up to 100 mM) with varying composition of organic solvents. Either acetonitrile or methanol or a mixture of the two are mostly utilized in the nonaqueous media. Primary considerations in developing NACE-MS are also discussed.

Acridines↗

PubMed1999

On-line sample clean-up and chromatography coupled with electrospray ionization mass spectrometry to characterize the primary sequence and disulfide bond content of recombinant calcium binding proteins.

We have used on-line sample clean-up, concentration, and chromatography with electrospray ionization mass spectrometry (ESI-MS), to characterize and determine the presence of disulfide bonds in recombinant full-length rat brain calbindin D28K and two deletion mutants of the protein, one lacking EF-hand 2 (calbindin delta 2) and the other lacking EF-hands 2 and 6 (calbindin delta 2,6). The molecular weights of the expressed proteins dissolved in biological buffers were determined with high accuracy using a low-flow, pressurized chamber infusion system, that allows on-line protein clean-up by removing buffers/salts incompatible with ESI-MS. The molecular weight determinations showed that the amino-terminal methionine residues had been cleaved during the expression and isolation of the recombinant proteins. Approximately 85-90% of the protein sequences were confirmed by on-line HPLC-ESI-MS analysis of peptides generated by a lysyl endoproteinase C digestion. Comparisons of ESI-MS spectra of native and reduced calbindin D28K and delta 2 show that the full length- and delta 2 mutant-protein contain one disulfide bond. Molecular mass determinations of calbindin delta 2,6 showed that this protein contains a highly active cysteine residue that covalently binds a mercaptoethanol group, or forms a homodimer via a disulfide bond. The results show surprising differences amongst the deletion mutants of calbindin D28K with respect to the formation of disulfide bonds. These differences are not readily detected by other techniques and show that ESI-MS is a powerful, rapid method by which to detect disulfide linkages for intact proteins.

Amino Acid Sequence↗

PubMed1999

Structural characterization of case-associated contaminants peak C and FF in L-tryptophan implicated in eosinophilia-myalgia syndrome.

We have characterized the structures of two case-associated contaminants of the Showa Denko L-tryptophan known to cause eosinophilia-myalgia syndrome (EMS). A combination of on-line accurate mass HPLC-electrospray ionization-mass spectrometry (LC-MS), HPLC-tandem mass spectrometry (LC-MS-MS) and HPLC-in source collision induced dissociation-MS-MS (LC-sCID-MS-MS) allowed the structure determination of both Peak C and FF. Peak C is identified as 3a-hydroxy-1,2,3,3a,8,8a-hexahydropyrrolo-[2-3b]-indole-2-carboxyl ic acid, whereas Peak FF is characterized as 2-(2-hydroxy-indoline)-tryptophan. Both contaminants contain indoline rings, and the significance of this finding is discussed.

Chromatography, High Pressure Liquid↗