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Biomedical subjects

K L Johnson

Publications and source records attributed to K L Johnson.

At least 55 records · Page 3Linked to original sources
PubMed1999

Eosinophilia-myalgia syndrome case-associated contaminants in commercially available 5-hydroxytryptophan.

Recently, 5-hydroxy-L-tryptophan (5-OHTrp) has been promoted as an alternative to banned L-tryptophan as a dietary supplement. It has been claimed to help alleviate obesity, insomnia, depression, and headaches. However, eosinophilia-myalgia syndrome (EMS)-like symptoms have also been associated with ingestion or exposure to 5-OHTrp. HPLC-UV analysis of EMS-implicated 5-OHTrp revealed the presence of peak X, described as case-implicated. We show that peak X is actually a family of contaminants with the same molecular weight (234 Da) and similar HPLC retention times. We also demonstrate that all eight samples of commercially available 5-OHTrp analyzed by HPLC-MS contained three or more contaminants of the peak X family. The significance of these findings is discussed.

5-Hydroxytryptophan↗
PubMed1999

Structural characterization of contaminants in commercial preparations of melatonin by on-line HPLC-electrospray ionization-tandem mass spectrometry.

Three different commercially available melatonin preparations were analyzed by on-line HPLC-electrospray ionization-tandem mass spectrometry. All three samples contained the same impurities at the approximately 0.1-0.5% level of parent melatonin. Based on accurate mass-HPLC-MS and tandem mass spectrometric analyses, two contaminants (both MH+ = 249) were identified as hydroxylation products of melatonin. One compound (MH+ = 265) was determined to be a C-2 oxidation product of hydroxymelatonin and a group of four regioisomers (MH+ = 477) were identified as melatonin-formaldehyde condensation products. These latter contaminants are structural analogues of the case-associated peak "E" found in L-tryptophan implicated in onset of eosinophilia-myalgia syndrome. The significance of these findings is discussed.

Chromatography, High Pressure Liquid↗
PubMed1999

Rapamycin inhibits didemnin B-induced apoptosis in human HL-60 cells: evidence for the possible involvement of FK506-binding protein 25.

In the present paper we show that the immunosuppressant rapamycin inhibits the induction of apoptosis by didemnin B in human promyeloid HL-60 cells. The mechanism of this inhibition is investigated using FK506, which competes with rapamycin for binding to their common target FK506-binding protein (FKBP)12. The lack of competition for rapamycin-mediated inhibition of didemnin B-induced apoptosis by FK506 suggests that rapamycin inhibits apoptosis through some mechanism other than inhibition of p70 S6 kinase activation. The lack of inhibition of didemnin B-induced apoptosis by inhibitors of phosphatidylinositol 3-kinase and mitogen-activated protein (MAP) kinase kinase further supports the conclusion that rapamycin does not inhibit didemnin B-induced apoptosis through inhibition of the MAP kinase pathway. Furthermore, didemnin B-induced apoptosis is not inhibited by the inhibitors of cyclin-dependent kinase, roscovitine and olomoucine. This indicates that rapamycin does not act through inhibition of cyclin-dependent kinases. Together with the lack of competition for the effect of rapamycin by FK506, our data suggest the possible involvement of the FK506-binding protein, FKBP25, which is localized in the nucleus. This interpretation of our data gains support from the fact that didemnin B does not induce apoptosis in enucleated HL-60 cells, which supports the possible involvement of FKBP25 in the inhibition of apoptosis by rapamycin.

Androstadienes↗
PubMed1999

Radiation-induced breakpoint misrejoining in human chromosomes: random or non-random?

PURPOSE: To investigate whether radiation-induced misrejoining of chromosome breakpoints is randomly or non-randomly distributed throughout the human genome. MATERIALS AND METHODS: Data were combined from as many published cytogenetic studies as possible. The percentage of radiation-induced breaks per megabase (Mb) of DNA between all human chromosomes was calculated, and the observed and expected numbers of breakpoints based on DNA content between and within chromosomes were compared. RESULTS: A DNA-proportional distribution of breakpoints in 14 autosomes and a statistically significant deviation from proportionality in the other eight autosomes and the sex chromosomes was found. Regression analysis showed no significant change in breakpoint frequency per Mb of DNA relative to autosome size. Analysis between chromosome arms showed a non-random distribution of induced breakpoints within certain autosomes, particularly the acrocentrics. In cases of non-random distributions, a prevalence of events was found at heterochromatic regions and/or telomeres, and a clustering of breakpoints was found near the centromeres of many chromosomes. CONCLUSIONS: There is an approximately linear proportionality between autosomal DNA content and observed breakpoint number, suggesting that subsets of autosomes can be used to estimate accurately the overall genomic frequency of misrejoined breakpoints contingent upon a carefully selected subset. However, this conclusion may not apply to the sex chromosomes. The results also support the influence of chromatin organization and/or preferential DNA repair/misrejoining on the distribution of induced breakpoints. However, these effects are not sufficient at a global level to dismiss the value of cytogenetic analysis using a genome subset for biodosimetry.

Chromosome Breakage↗
PubMed1999

Functional coupling between replication and packaging of poliovirus replicon RNA.

Poliovirus RNA genomes that contained deletions in the capsid-coding regions were synthesized in monkey kidney cells that constitutively expressed T7 RNA polymerase. These replication-competent subgenomic RNAs, or replicons (G. Kaplan and V. R. Racaniello, J. Virol. 62:1687-1696, 1988), were encapsidated in trans by superinfecting polioviruses. When superinfecting poliovirus resistant to the antiviral compound guanidine was used, the RNA replication of the replicon RNAs could be inhibited independently of the RNA replication of the guanidine-resistant helper virus. Inhibiting the replication of the replicon RNA also profoundly inhibited its trans-encapsidation, even though the capsid proteins present in the cells could efficiently encapsidate the helper virus. The observed coupling between RNA replication and RNA packaging could account for the specificity of poliovirus RNA packaging in infected cells and the observed effects of mutations in the coding regions of nonstructural proteins on virion morphogenesis. It is suggested that this coupling results from direct interactions between the RNA replication machinery and the capsid proteins. The coupling of RNA packaging to RNA replication and of RNA replication to translation (J. E. Novak and K. Kirkegaard, Genes Dev. 8:1726-1737, 1994) could serve as mechanisms for late proofreading of poliovirus RNA, allowing only those RNA genomes capable of translating a full complement of functional RNA replication proteins to be propagated.

Base Sequence↗
PubMed1999

Induction and maintenance of autonomous flock house virus RNA1 replication.

The nodavirus flock house virus (FHV) has a bipartite, positive-sense, RNA genome that encodes the catalytic subunit of the RNA replicase and the viral capsid protein precursor on separate genomic segments (RNA1 and RNA2, respectively). RNA1 can replicate autonomously when transfected into permissive cells, allowing study of the kinetics of RNA1 replication in the absence of either RNA2 or capsid proteins. However, RNA1 replication ceases ca. 3 days after transfection despite the presence of replication-competent RNA. We examined this inhibition by inducing the expression of RNA1 in cells from a cDNA copy that was under the control of a hormone-regulated RNA polymerase II promoter. This system reproduced the shutoff of RNA replication when DNA-templated primary transcription was turned off. Continued primary transcription partially alleviated the shutoff and maintained the rate of RNA replication for several days at a steady-state level approximately one-third that of the peak rate. After shutoff, RNA replication could be restored by transferring the resulting intracellular RNA to fresh cells or by reinducing primary transcription, indicating that cessation of replication occurred despite the competence of both the viral RNA and the cytoplasmic environment. These data suggest that there is a mechanism by which replication is shut off at late times after transfection, which may reflect the natural endpoint of the replicative cycle.

Animals↗
PubMed1999

Subglottic stenosis: correlation between computed tomography and bronchoscopy.

The evaluation of subglottic stenosis has been limited by the lack of standardized methods for determining the cross-sectional area and length of the stenotic segment. A rabbit model was used to prospectively evaluate the correlation between computed tomography (CT) and bronchoscopy in the evaluation of this disease. Subglottic stenosis was produced in 39 New Zealand White rabbits by a transoral endoscopic technique. The animals were evaluated 3 weeks later with spiral CT, rigid bronchoscopy, and open laryngotracheal exploration. Spiral CT was performed with the location, degree, and length of subglottic stenosis being determined by a blinded observer. Each animal then underwent rigid bronchoscopy and open laryngotracheal exploration for determination of the same measurements. Data were analyzed to determine the correlation between the radiographic and surgical techniques in evaluating the airway stenosis. With regard to the degree of stenosis, 94% of the rabbits were determined to have CT and bronchoscopic measurements that were within 15% (Pearson correlation .94, p < .05). With regard to the length of stenosis, 94% of animals had a measurement on CT that was within 2 mm of that observed upon open exploration (Pearson correlation .81, p < .05). The CT evaluation of subglottic stenosis correlated well with the currently used method of visual inspection at bronchoscopy in evaluating tracheal stenosis in this animal model. These data suggest that CT could serve as a useful adjunct in the evaluation of tracheal stenosis, especially when serial examinations are required.

Animals↗
PubMed1999

Chromosome aberrations of clonal origin in irradiated and unexposed individuals: assessment and implications.

Chromosome painting has proven useful for the detection of chromosomal rearrangements, although the presence of cells containing clonal aberrations can have an effect on the outcome of cytogenetic analyses (e.g. aberration frequency and chromosomal distribution studies). Cells with clonal chromosomal changes have been found in studies of both radiation-exposed Chernobyl cleanup workers ("liquidators") and healthy unexposed human subjects. We have used a simple statistical method to aid in the identification of individuals from distinct Chernobyl radiation-exposed and unexposed control populations who may possess cells containing clonal rearrangements. A chi2 value determined from the observed number of aberrations and the expected number based on chromosome length that corresponds to a probability less than 0.005 appears to be an indicator of clonality. These selected individuals can be analyzed further for clonality, thereby sparing detailed examination of the entire population. Here we present an analysis of individuals possessing clonal aberrations to assess the influence of clonality on the results of cytogenetic studies. Our results show that the subtraction of clonal events from the chi2 calculation for the "outliers" results in nearly all of these values losing their statistically significant deviation from proportionality. These adjustments can also be made to prevent the overestimation of frequencies of chromosome aberrations for biodosimetry. The frequency of clonal aberrations appears to increase as a function of age in control subjects, whereas an age effect was not evident in Chernobyl liquidators. This suggests that spontaneous and radiation-induced clonal expansion are occurring in control subjects and liquidators, respectively.

Adult↗
PubMed1999

Therapeutic paralysis of critically ill trauma patients: perceptions of patients and their family members.

BACKGROUND: Neuromuscular blocking agents are used in critically ill patients to induce therapeutic paralysis. These drugs leave patients fully immobile but conscious. Analgesics and sedatives are concomitantly administered with the paralytic agents. Little is known about what patients remember when they receive these combinations of drugs. Even less is known about the experiences and needs of the patients' family members during the patients' paralysis. OBJECTIVES: To obtain recollections of therapeutic paralysis in critically ill adult trauma patients and to determine the psychological, emotional, and educational needs of the patients' family members during the time the patients were paralyzed. METHODS: A qualitative phenomenological approach was used to investigate the "lived" experience of therapeutic paralysis of 11 pairs of subjects. Each pair consisted of one critically ill adult trauma patient and one member of the patient's family. RESULTS: The patients recalled their experience of therapeutic paralysis with vagueness, as if they had been dreaming. Few recalled pain or painful procedures. Patients remembered having nurses and family members provide emotional support and encouragement. Family members understood the rationale for use of the drugs. They remembered being encouraged to touch and talk with patients. The subjects suggested providing additional education about events that occur when paralysis is being reversed. CONCLUSIONS: Two confounding variables may have affected these findings: a liberalized visiting policy and use of effective pain and sedation protocols. Healthcare professionals and patients' family members should monitor bedside conversations and use touch and words of encouragement to support patients during therapeutic paralysis.

Adaptation, Psychological↗
PubMed1998

On-line HPLC-tandem mass spectrometry structural characterization of case-associated contaminants of L-tryptophan implicated with the onset of eosinophilia myalgia syndrome.

The eosinophilia-myalgia syndrome (EMS) outbreak that occurred in the USA in 1989 was caused by the intake of L-tryptophan (Trp) produced from one manufacturer, Showa Denko K.K. of Japan. Six compounds present in the Trp were reported to be case-associated contaminants. However, three of these compounds, Peaks C, FF and AAA have remained unidentified. Here, we successfully employ on-line HPLC-electrospray ionization multistage mass spectrometry to structurally characterize Peak C and Peak FF. Peak C was determined by accurate mass-LC-MS to have a protonated molecular ion MH+ = 221.0919 with an empirical formula of C11H13N2O3. By comparing the LC-MS-MS spectra with authentic 5-OHTrp and other structurally similar compounds, as well as considering the chemical reactivity of the indole ring, the structure of Peak C was consistent with 3a-hydroxy-1,2,3,3a,8,8a-hexahydropyrrolo-[2,3-b]-indole-2-carboxy lic acid. Peak FF was also subjected to accurate mass-LC-MS and shown to have MH+ = 338.1524, corresponding to an empirical formula of C19H20N3O3. Comparison of the LC-MS-MS and LC-sCID-MS-MS of spectra derived from Peak FF with a previously characterized contaminant of Trp, namely P31, was consistent with Peak FF being 2-(2-hydroxy indoline)-Trp. Unlike the majority of the contaminants identified in EMS implicated tryptophan, both Peaks C and FF possess an indoline ring. This is significant since a case-associated contaminant found in 5-hydroxy-Trp also contains an indoline ring, and the chemical reactivity of this ring system may possibly play a role in the etiology of EMS.

Chromatography, High Pressure Liquid↗
PubMed1998

7-Oxo-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]pyridines as novel inhibitors of human eosinophil phosphodiesterase.

High-throughput file screening against inhibition of human lung PDE4 led to the discovery of 3-ethyl-1-(4-fluorophenyl)-6-phenyl-7-oxo-4, 5,6,7-tetrahydro-1H-pyrazolo[3,4-c]pyridine (11) as a novel PDE4 inhibitor. Subsequent SAR development, using an eosinophil PDE assay, led to analogues up to 50-fold more potent than 11 with IC50 values of 0.03-1.6 microM. One such compound, CP-220,629 (22) (IC50 = 0.44 microM), was efficacious in the guinea pig aerosolized antigen induced airway obstruction assay (ED50 2.0 mg/kg, po) and demonstrated a significant reduction in eosinophil (55%), neutrophil (65%), and IL-1beta (82%) responses to antigen challenge in atopic monkeys (10 mg/kg, po).

Airway Obstruction↗
PubMed1998

Enhanced sensitivity for sequence determination of major histocompatibility complex class I peptides by membrane preconcentration-capillary electrophoresis-microspray-tandem mass spectrometry.

Sequence analysis of antigenic major histocompatibility complex (MHC) class I peptides requires minimizing sample loss and enhancing mass spectrometric sensitivity. In order to facilitate such analyses, we have coupled on-line membrane preconcentration-capillary electrophoresis (mPC-CE) with microspray mass spectrometry (mPC-CE-microMS) and tandem mass spectrometry (mPC-CE-microMS/MS). Specifically, cell lysate from approximately 10(9) EG-7 mouse tumor cells was immunoprecipitated and the released MHC class I peptides were subjected to reverse-phase HPLC. An HPLC fraction containing antigenic peptide(s) shown to induce T-cell stimulation was subjected to mPC-CE-microMS. Approximately 10 microL (from 100 microL) of the fraction was pressure-injected and concentrated on a styrenedivinylbenzene (SDB) impregnated membrane. The peptides were eluted from the membrane with approximately 100 nL of 80% methanol, sandwiched between a leading stacking buffer (LSB, also serving as CE separation medium) of approximately 110 nL of 0.1% acetic acid in 10% methanol, and a trailing stacking buffer (TSB) of approximately 110 nL of 0.1% NH4OH. On application of the CE voltage the peptides are subjected to moving boundary transient isotachophoresis and focused. The peptides were separated in a Polybrene-coated capillary with application of -20 kV in reverse polarity mode and subsequently sprayed via an emitter coupled to the CE capillary by a liquid junction containing a platinum wire. An ion at m/z 482.3 was detected and subjected to mPC-CE-microMS/MS and determined to be SIINFEKL, a peptide (OVA) known to be antigenic in the mouse model system. Sensitivity enhancement over conventional mPC-CE-MS and MS/MS was approximately 100-fold.

Animals↗
PubMed1998

Zinc-induced conformational changes in the DNA-binding domain of the vitamin D receptor determined by electrospray ionization mass spectrometry.

Electrospray ionization mass spectrometry (ESI-MS) was used to measure conformational changes within the DNA-binding domain of the vitamin D receptor (VDR DBD) upon binding zinc (Zn2+). As increasing concentrations of Zn2+ were added to the VDR DBD, a gradual shift in the mass envelope to lower charge states was observed in the multiply charged spectrum. The shift in the charge states was correlated to changes observed in the far-ultraviolet circular dichroic (far-UV CD) spectrum of the protein as it was titrated with Zn2+. Both the multiply charged ESI and far-UV CD spectra of the Zn(2+)-titrated protein show that the binding of the first Zn2+ ion to the protein results in very little conformational change in the protein. The binding of a second Zn2+ ion resulted in a significant alteration in the structure of the protein as indicated by changes in both the multiply charged ESI and far-UV CD spectra. Much smaller changes were seen within the multiply charged ESI or far-UV CD spectra upon increasing the Zn2+ concentration beyond 2 mol/mol of protein. The results presented indicate that ESI-MS in combination with CD is a powerful method to measure gross conformational changes induced by the binding of metals to metalloproteins.

Circular Dichroism↗
PubMed1998

Role of nitric oxide in kainic acid-induced elevation of cochlear compound action potential thresholds.

Nitric oxide (NO) has specifically been found to mediate the effects of excitatory amino acids in the central nervous system (CNS). Excitatory amino acids are the primary neurotransmitters at the cochlear hair cell afferent nerve synapse. Recent studies in our laboratory demonstrate that NO synthase is an active enzyme in the spiral ganglion cells of the cochlea. Given our current understanding of neurotransmission in the cochlea, it is reasonable to postulate that the actions of NO in cochlear neuronal tissue are similar to the actions of NO in the CNS, and that NO acts as a neurotransmitter/neuromodulator in the cochlea. In addition, NO is implicated as a mediator of excitotoxicity in the CNS and may therefore play a similar role in excitotoxicity in the cochlea. To further elucidate the role of NO in cochlear excitotoxicity, this study investigated the effects of 7-nitroindazole (7-NI), a competitive inhibitor of neuronal nitric oxide synthase, with regard to kainic acid (KA)-induced elevation of compound action potential (CAP) thresholds. KA is a conformationally restricted analog of glutamate with well-known excitotoxic effects on SGC's and previously described inhibitory actions on cochlear CAP thresholds. In anesthetized gerbils, CAP thresholds were recorded before and after cochlear perfusions with control solutions of artificial perilymph solution and test solutions of KA. 7-NI was administered i.p. prior to KA perfusion in an effort to block its depolarizing and toxic effects. Results showed that cochlear perfusion with KA caused significant elevation (p < 0.05) of the mean CAP threshold. This threshold shift was significantly reduced (p < 0.05) in animals pretreated with 7-NI. These results indicate that NO is involved in the toxic effects on CAP thresholds elicited by KA in the cochlea.

Action Potentials↗
PubMed1998

Frequency, distribution and clonality of chromosome damage in human lymphocytes by multi-color FISH.

Whole chromosome painting was used to determine whether the use of different sets of paints would influence results obtained from the analysis of human peripheral blood lymphocytes from 27 healthy unexposed subjects. Painting was also used to determine if aberration frequencies are proportional to the size of selected chromosomes in human lymphocytes irradiated in vitro. The in vitro results showed that the frequencies of radiation-induced stable aberrations (i.e. translocations and insertions) in chromosomes 3, 5 and 6 painted in unique colors are proportional to chromosome size. Aberration frequencies in the normal subjects were measured using two different sets of paints, one set for chromosomes 3, 5 and 6 where each chromosome was labeled in a unique color and one set where chromosomes 1, 2 and 4 were painted in a single color. The frequency of aberrations among chromosomes 1-6 in the population as a whole was also found to be proportional to chromosome size. However, some individual subjects had a distribution of damage that was not proportional to chromosome size due to the presence of clones of abnormal cells. Aberration frequencies measured in chromosomes 3, 5 and 6 as a set were highly correlated with those observed in chromosomes 1, 2 and 4 as a set, after adjusting for the different amounts of the genome that were painted. We also determined whether differences exist in the aberration frequencies measured by two scoring systems: the classical method, where reciprocal exchanges are scored as single events, and PAINT, where each break junction is scored as a single event. The two scoring systems gave highly correlated results for translocations and differed by a constant value (PAINT x 0.58 = classical method). Approximately 27% of translocations were observed to be non-reciprocal due to a failure to detect exchanges involving small amounts of material or to a non-reciprocal exchange mechanism. Our results support the hypothesis that cytogenetic evaluations for biodosimetry can be performed with any one or more of the chromosomes studied here and indicate that the aberration frequency measurements are independent of the scoring system selected for the evaluation. We also present a simple statistical method for identifying subjects that may possess clonal aberrations.

Adolescent↗