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Biomedical subjects

K L Johnson

Publications and source records attributed to K L Johnson.

At least 73 records · Page 4Linked to original sources
PubMed1998

Fluorescence in situ hybridization (FISH): DNA probe production and hybridization criteria.

We describe methods for the production of fluorescence in situ hybridization (FISH) probes and the utilization of these probes for the detection of complementary DNA sequences with accuracy and sensitivity for application in both basic research and clinical diagnosis. Due to the frequent use of FISH in many laboratories, it is important to apply the most convenient and reproducible approach. This review describes some of the most recent techniques, and includes versatile, effective and simple methods of probe production and fluorescence in situ hybridization. We also describe methods for the production of region-specific and chromosome-specific DNA probes and hybridization techniques for the visualization of these probes.

Animals↗
PubMed1997

Inhibition of human mitochondrial aldehyde dehydrogenase by the disulfiram metabolite S-methyl-N,N-diethylthiocarbamoyl sulfoxide: structural characterization of the enzyme adduct by HPLC-tandem mass spectrometry.

S-Methyl-N,N-diethylthiocarbamoyl sulfoxide (MeDTC-SO) is a known metabolite of the aversion therapy drug disulfiram (DSF). MeDTC-SO is also a potent inhibitor of human mitochondrial aldehyde dehydrogenase (hmALDH) with an IC50 of 1.5 microM. Inhibition of the enzyme by MeDTC-SO resulted in the addition of approximately 100 Da to the molecular mass of the intact protein, as determined by on-line HPLC-electrospray ionization MS (LC-MS). Dialysis of the inhibited protein did not reverse the inhibition, and the molecular mass of 54,533 Da (+/- 0.01%) remained unchanged, indicating that a covalent modification of the protein had occurred. Proteolytic digestion of hmALDH under basic conditions using trypsin at pH 7.8 revealed that the adduct was base labile. However, treating the adducted protein with endopeptidase-Glu-C at pH 3.7 produced a peptide adduct at MH+ = 4924, tentatively attributable to a carbamoylated peptide. This peptide contains three adjacent cysteines, one of which has been implicated as a key amino acid in the highly conserved active site region of ALDH. A pepsin digestion of hmALDH carried out at pH 3.7 and subsequent LC-MS analysis revealed an ion at MH2(2+) = 501.5, corresponding to the carbamoylated peptide FNQGQC1C2C3. This peptide contains the same adjacent active site cysteines. This latter peptide was subjected to LC-MS/MS, which enabled us to determine that the site of carbamoylation was at Cys2. The MS/MS product ion data also confirmed the presence of a carbamoyl group as the adduct species.

Aldehyde Dehydrogenase↗
PubMed1997

Zinc binding properties of the DNA binding domain of the 1,25-dihydroxyvitamin D3 receptor.

To assess the zinc binding stoichiometry and the structural changes induced upon the binding of zinc to the human vitamin D receptor (VDR), we expressed the DNA binding domain (DBD) of the human VDR in bacteria as a soluble glutathione-S-transferase fusion protein at 20 degrees C, and examined the apo-protein and metal-liganded protein by mass spectrometry, and circular dichroism and nuclear magnetic resonance spectroscopy. Following final preparation with a zinc-free buffer, the VDR DBD bound 2 mol of zinc/mol of protein as measured by inductively coupled plasma-mass spectrometry and electrospray ionization-mass spectrometry. When protein preparation was carried out in a zinc containing buffer and zinc content of the protein was assesed by the same methods, VDR DBD bound 4 mol of zinc/mol of protein. Analysis of the protein using circular dichroism spectroscopy demonstrated that the EDTA-treated protein increased in alpha-helical content from 16 to 27% on the addition of zinc. Equilibrium ultracentrifugal analyses of the VDR DBD indicated that the protein was present in solution as a monomer. Gel mobility shift analyses of the VDR DBD with several vitamin D response elements (VDREs) in the absence of accessory proteins such as retinoic acid receptor, showed that VDR DBD was able to form a protein/VDRE DNA structural complex. In the presence of zinc, proton NMR NOESY spectra showed that the protein possessed elements of secondary structure. The addition of VDRE DNA, but not random DNA, caused changes in the proton NMR spectra of VDRE DNA indicating specific interaction between protein and DNA groups. We conclude that the DBD of the VDR binds zinc and DNA and undergoes conformational changes on binding to the metal and DNA.

Amino Acid Sequence↗
PubMed1997

Determination of calcium-binding sites in rat brain calbindin D28K by electrospray ionization mass spectrometry.

Calbindin D28K, a member of the troponin-C superfamily of calcium-binding proteins, contains six putative EF-hand domains. Calcium-binding studies of the protein by different groups of investigators have yielded discordant results with respect to the stoichiometry of calcium-binding. It has been suggested that the protein binds anywhere from 3-6 mol of calcium/mol of protein. We used negative ion electrospray ionization mass spectrometry in order to definitively determine the exact calcium-binding stoichiometry of calbindin D28K and two mutant forms of the protein, one lacking EF-hand 2 (delta2) and the other lacking EF-hands 2 and 6 (delta2,6). The full-length protein bound 4 mol of calcium/mol of protein, while both of the deletion mutants bound 3 mol of calcium. Since terbium has been used extensively as a probe for the determination of the calcium-binding stoichiometries of calcium-binding proteins, we also examined the binding of terbium to the three proteins under the same conditions. Full-length calbindin D28K bound 4 mol of terbium/mol of protein, while calbindin delta2 and delta2,6 each bound 3 mol. These results clearly show that calbindin D28K binds 4 mol of calcium/mol of protein and that terbium-binding stoichiometry is similar to that of calcium.

Amino Acid Sequence↗
PubMed1997

Inhibition of aldehyde dehydrogenase by disulfiram and its metabolite methyl diethylthiocarbamoyl-sulfoxide.

Disulfiram (DSF) is presently the only available drug used in the aversion therapy of recovering alcoholics. It acts by inhibiting aldehyde dehydrogenase (ALDH), leading to high blood levels of acetaldehyde. The in vitro inhibition of ALDH by DSF and its metabolites was systematically studied by combined enzyme inhibition assay with direct molecular weight determination of the same sample using electrospray ionization-mass spectrometry (ESI-MS). Enzyme activity was measured after incubating yeast ALDH (yALDH) with excess concentrations of DSF, methyl diethyldithiocarbamate (MeDDC) and methyl diethylthiocarbamoyl-sulfoxide (MeDTC-SO) and then subjected to analysis by ESI-MS. Addition of DSF resulted in complete enzyme inhibition; however, ESI-MS analysis demonstrated no discernible shift in molecular weight, indicating that no intermolecular adduct was formed with the protein. Treatment of yALDH with MeDTC-SO also completely abolished yALDH activity with a concomitant increase of + approximately 100 Da in the molecular mass of the enzyme. This indicated formation of a covalent carbamoyl protein adduct. Furthermore, the effects of dithiothreitol (DTT) were examined on samples of inhibited protein in vitro. At pH 7.5, DTT completely reversed inhibition after DSF treatment. yALDH inhibited by MeDTC-SO could not be recovered by DTT at pH 7.5, but at pH 9 the enzymic activity was fully restored and a mass loss of approximately 100 Da was noted. This observations are consistent with mechanisms where inhibition of yALDH by DSF in vitro involves oxidation of the active site, whereas MeDTC-SO forms a covalent adduct with the protein in vitro resulting in cessation of enzyme activity.

Aldehyde Dehydrogenase↗
PubMed1997

Replication of flock house virus RNAs from primary transcripts made in cells by RNA polymerase II.

To develop vector systems that combine high transcription activity with biologically safe delivery vehicles, we have explored the use of RNA replication to amplify mRNAs, by using flock house virus (FHV) as a model system. The FHV RNA replicase is encoded in the larger of the two segments that comprise the viral positive-sense RNA genome. A cDNA copy of this self-replicating RNA was precisely positioned between a promoter site for cellular RNA polymerase II and a cDNA encoding a self-cleaving ribozyme from hepatitis delta virus. Transfection of this plasmid into cultured BHK cells resulted in prolonged, autonomous FHV RNA replication in the cytoplasm and substantial amplification of the RNA replicon. The replicase also amplified RNA transcribed from a second plasmid of similar design that contained a cDNA copy of the other FHV genome segment. These results constitute a significant step toward the harnessing of nodaviral RNA replication as the basis of a versatile vector system.

Animals↗
PubMed1997

Promotion of endometriosis in mice by polychlorinated dibenzo-p-dioxins, dibenzofurans, and biphenyls.

Previous studies showed exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) enhances the development of endometriotic lesions. In this study we examined the effects of other polyhalogenated aromatic hydrocarbons on endometriotic proliferation. B6C3F1 female mice were treated via oral gavage a total of five times, with 3 weeks between each dosing, with 0, 1, 3, or 10 micrograms 2,3,7,8,-TCDD/kg body weight (bw); 3 or 30 mg 2,2',4,4',5,5'-hexachlorobiphenyl (PCB 153)/kg bw; 100, 300, or 1000 micrograms 3,3',4,4',5-pentachlorobiphenyl (PCB 126)/kg bw; 10, 30, or 100 micrograms 2,3,4,7,8-pentachlorodibenzofuran (4-PeCDF)/kg bw; or 2 or 20 mg 1,3,6,8-TCDD/kg at 10 ml/kg bw. Endometriosis was surgically induced during the week of the second dosing. Three weeks following the final dose, the mice were euthanized and endometriotic lesions, whole body, liver, ovaries, uterine horn, and thymus were weighted, and lesion diameters were measured. Lesions, uterine horns, and ovaries were fixed for histopathology and livers were processed for measurement of ethoxyresorufin O-deethylase (EROD) activity. Both 2,3,7,8-TCDD (1 and 3 micrograms/kg bw) and 4-PeCDF (100 micrograms/kg bw) significantly enhanced the growth of endometrial lesions. No statistically significant increase in endometriotic lesion size was detected in animals treated with either PCB 126 or with the highest dose of 2,3,7,8-TCDD, possibly due to the effects of histologically observed ovarian toxicity. The nondioxin-like compounds, PCB 153 and 1,3,6,8-TCDD, produced no observable effects on endometriosis. Hepatic EROD activity was significantly induced by 2,3,7,8-TCDD, 4-PeCDF, and PCB 126, but not by PCB 153 or 1,3,6,8-TCDD. The results of this study provide preliminary support for the hypothesis that halogenated aromatic hydrocarbon-promoted endometriosis may be Ah receptor mediated.

Animals↗
PubMed1997

The use of environmental control units by occupational therapists in spinal cord injury and disease services.

OBJECTIVES: This study investigated issues related to the use of environmental control units (ECUs) with persons who have spinal cord injury and disease (SCI/D) to ascertain what, if any, changes are needed to optimize occupational therapy service delivery in this area. METHOD: A questionnaire was mailed to 120 occupational therapists employed by facilities nationwide that serve person with SCI/D. The survey addressed the occupational therapist's role in the recommendation and use of ECUs, funding sources, and training needs. The response rate was 89% (n = 107). RESULTS: Most respondents (88%) indicated that occupational therapists were the predominant professionals in their facilities who evaluated and recommended ECUs. The majority of respondents (84%) reported using ECUs in hospital-based treatment with persons with SCI/D. However, respondents only recommended ECUs for home use for fewer than 25% of their clients. High cost and the lack of third-party reimbursement were the primary reasons that deterred respondents from recommending ECUs for home use. Fifty-five percent of the respondents reported a need for basic training and 86% for more in-depth education and training in environmental control technologies. CONCLUSION: Outcomes data are needed to support the use of ECUs and to educate third-party payers about the benefits of ECUs for clients with quadriplegia in order to increase rates of reimbursement. In addition, occupational therapists themselves may need improved training in evaluating and training clients in the use of ECU technology.

Communication Devices for People with Disabilities↗
PubMed1996

Protein tyrosine kinase inhibitors prevent didemnin B-induced apoptosis in HL-60 cells.

Didemnin B induces rapid apoptosis in human promyeloid HL-60 cells with an optimal concentration of 1 microM (Grubb et al. (1995) Biochem. Biophys. Res. Commum. 215, 1130-1136), but little is known about how it does so. In order to determine whether protein tyrosine phosphorylation is involved in this rapid induction of apoptosis, HL-60 cells were pre-treated with tyrosine kinase inhibitors for 1 h before didemnin B treatment. Genistein, 2,5-dihydroxycinnamic acid methyl ester, and a range of tyrphostins inhibit didemnin B-induced apoptotic morphology in a concentration-dependent manner. DNA fragmentation induced by didemnin B is also inhibited by genistein, 2,5-dihydroxycinnamic acid methyl ester, and tyrphostins.

Antineoplastic Agents↗
PubMed1996

Subcellular localization of TCDD differs between the liver, lungs, and kidneys after acute and subchronic exposure: species/dose comparisons and possible mechanism.

Subcellular localization of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds has been examined only in the liver. The objective of this study was (1) to examine and compare the subcellular distribution of TCDD within hepatic and nonhepatic (lungs/kidneys) tissues of female Sprague-Dawley rats acutely exposed to TCDD, (2) to analyze species comparisons in the subcellular localization of TCDD in multiple tissues, (3) to investigate the effect of dose on subcellular distribution of TCDD, (4) to analyze the effect of subchronic exposure on the subcellular distribution of TCDD, and (5) to examine one possible mechanism for subcellular localization of TCDD. Female Sprague-Dawley rats and B6C3F1 mice received a single oral dose of 0.1, 1.0, or 10 microg [3H]TCDD/kg body weight and subcellular fractions of the liver, lungs, and kidneys were prepared by differential centrifugation 3 days after exposure. Analysis of the rat subcellular fractions revealed that TCDD was equally distributed between the hepatic P9 (mitochondrial, lysosomal, and nuclear) and S9 (cytosol and microsomal) fractions at all doses tested. In contrast, TCDD was concentrated in the P9 of rat nonhepatic tissues at all doses studied. Differential centrifugation of the hepatic S9 showed that TCDD was localized within the hepatic P100 (microsomal) fraction at all doses tested. In contrast, TCDD localized in pulmonary and renal S100 (cytosolic) fractions at all doses. The subcellular distribution of TCDD in the liver and lungs of acutely exposed B6C3F1 mice was similar to that observed in the rats. Although TCDD was concentrated within the renal P9, the remainder of TCDD in the S9 was evenly distributed between the S100 and the P100 fractions of acutely exposed B6C3F1 mice. Subchronic exposure of B6C3F1 mice to 1.5 or 150 ng [3H]TCDD/kg/day revealed that increasing dose resulted in equal distribution of TCDD between the hepatic S9 and P9 versus concentration in the renal P9. In addition, a dose-dependent increase in accumulation of TCDD in the hepatic P100 was accompanied by a dose-dependent increase in TCDD localization in the renal S100 of mice subchronically exposed to TCDD. TCDD exposure in rats resulted in a dose-dependent increase in the induction of CYP1A1 protein and associated enzyme activity in hepatic, pulmonary, and renal microsomes. TCDD-induced CYP1A2 protein levels and associated enzymatic activity were only present in hepatic microsomes. This is the first report to suggest that subcellular distribution of TCDD differs between hepatic and nonhepatic tissues and demonstrate that the liver-specific microsomal localization of TCDD in female Sprague-Dawley rats also occurs in the liver of female B6C3F1 mice acutely or subchronically exposed to TCDD. In addition, these data are consistent with the hypothesis that the hepatic sequestration of TCDD is due to an interaction with CYP1A2. Furthermore, the lack of pulmonary/renal sequestration coupled with the lack of localization of TCDD in pulmonary/renal microsomes also supports the role of CYP1A2 as a hepatic microsomal binding protein involved in TCDD sequestration..

Animals↗
PubMed1995

Nocturnal blood pressure measurement: can it be predicted by self-monitoring and is 24-hr BP monitoring clinically important in the treatment of hypertension.

We predict that 24-hour blood pressure data will be required prior to starting treatment in order to receive third-party payment. We are currently performing a cost benefit analysis in patients with mild to moderate hypertension to see the prevalence of inappropriate treatment of hypertension (diagnosed by normal 24-hour BP results) in an outpatient clinic population. We will compare the cost of treating that segment of the population that is inappropriately on treatment to the cost of 24-hour BP monitoring for the entire study population. As a result of this type of analysis, health care payors may require 24-hour BP documentation prior to hypertension treatment for all cases of mild to moderate hypertension.

Blood Pressure Determination↗
PubMed1994

Photolysis of sulfiram: a mechanism for its disulfiram-like reaction.

Sulfiram, a drug applied topically to treat scabies, produces effects similar to those of disulfiram after subsequent ingestion of ethanol. Disulfiram, used in aversion therapy in the treatment of alcoholism, inhibits hepatic aldehyde dehydrogenase (ALDH) causing an accumulation of acetaldehyde after ethanol ingestion. The increased tissue levels of acetaldehyde cause a spectrum of undesirable side-effects including flushing, nausea, vomiting, and tachycardia, which are referred to as the disulfiram reaction. Previous studies have shown that in vitro sulfiram is a very weak inhibitor of ALDH, but solutions of sulfiram markedly increase in potency with time. In the present study, fresh solutions of sulfiram were exposed to fluorescent room light under ambient conditions and analyzed at timed intervals by HPLC. At least eight products, including disulfiram, were formed in the light-exposed sulfiram solutions, but not in solutions kept in the dark. Structural characterization of two of the photolysis products was obtained by on-line microbore HPLC-mass spectrometry (mu LC-MS) and on-line microbore HPLC-tandem mass spectrometry (mu LC-MS/MS) using continuous flow-liquid secondary ion mass spectrometry (CF-LSIMS) as the primary ionization method. Sulfiram was converted to disulfiram at an initial rate of 0.7%/hr, and the formation of disulfiram correlated with the increase in ALDH inhibition in vitro. The results of this investigation show that while sulfiram is a weak inhibitor of ALDH in vitro, it is readily photoconverted to disulfiram, a very potent inhibitor of ALDH, which may explain the adverse reaction to ethanol after sulfiram therapy.

Aldehyde Dehydrogenase↗
PubMed1994

Mitochondrial respiratory chain inhibitors induce apoptosis.

In this paper the specific mitochondrial respiratory chain inhibitors rotenone and antimycin A and the highly specific mitochondrial ATP-synthase inhibitor oligomycin are shown to induce an apoptotic suicide response in cultured human lymphoblastoid and other mammalian cells within 12-18 h. The mitochondrial inhibitors do not induce apoptosis in cells depleted of mitochondrial DNA and thus lacking an intact mitochondrial respiratory chain. Apoptosis induced by respiratory chain inhibitors is not inhibited by the presence of Bcl-2. We discuss the possible role of mitochondrial induced apoptosis in the ageing process and age-associated diseases.

Animals↗
PubMed1994

Investigation of drug metabolism using capillary electrophoresis with photodiode array detection and online mass spectrometry equipped with an array detector.

The application of capillary electrophoresis (CE) with photodiode array detection (DAD) and on-line CE-mass spectrometry (CE-MS) equipped with a position and time resolved (PATRIC) focal plane detector for analysis of both in vitro and in vivo drug metabolism is demonstrated. Separation of metabolites derived from the neuroleptic drug haloperidol, by CE, using a simple, volatile run buffer containing 50 mM ammonium acetate with 10% methanol and 1% acetic acid is reported. The potential utility of CE-DAD for screening drug metabolite mixtures derived from hepatic microsomal incubations is demonstrated for haloperidol (HAL). Also the potential problems associated with using this technology to screen human urine samples for HAL metabolites is discussed. Furthermore, the usefulness of CE-MS and CE-electrospray ionization skimmer collision induced dissociation-MS (CE-ESI-CID-MS) in identification and structure elucidation of HAL metabolites derived from both a guinea pig hepatic microsomal incubation and urine from a patient treated with 0.5 mg/day of HAL is shown. The utility of such an approach in the general area of clinical pharmacology is also discussed.

Animals↗
PubMed1994

Sulfonamide-reactive lymphocytes detected at very low frequency in the peripheral blood of patients with drug-induced eruptions.

BACKGROUND: The role of T lymphocytes in mediating drug eruptions is uncertain. METHODS: Twenty-four patients with eruptions induced by sulfonamide-related drugs were studied to detect lymphocyte reactivity to drugs. Both the lymphocyte transformation test and limiting dilution analysis were used as assays for drug-reactive lymphocytes. Peripheral blood lymphocytes were expanded in interleukin-2 and tested for reactivity to sulfamethoxazole and furosemide. RESULTS: The lymphocyte transformation test results to sulfamethoxazole, sulfisoxazole, and furosemide were found to be generally unreliable with a high rate of false-negative and false-positive results. However, as determined by limiting dilution analysis, sulfamethoxazole-reactive lymphocytes were detected in the peripheral blood of one patient at a frequency of 1/172,000. This is within the lower range of frequencies of urushiol-reactive T cells in the peripheral blood of patients with allergic contact dermatitis to urushiol (poison ivy). Two sulfonamide-reactive lymphocyte lines were cultured from two patients. Both lines proliferated in response to sulfamethoxazole but not in response to furosemide, suggesting that furosemide does not cross-react with the sulfonamides. CONCLUSIONS: Lymphocytes reactive to sulfamethoxazole were detected at low frequencies in the peripheral blood of three patients with drug eruptions secondary to administration of sulfamethoxazole.

Adult↗
PubMed1994

Closed versus open endotracheal suctioning: costs and physiologic consequences.

OBJECTIVE: To examine the physiologic consequences and costs associated with two methods of endotracheal suctioning: closed vs. open. DESIGN: A prospective, randomized, controlled study. SETTING: An eight-bed trauma intensive care unit (ICU) in a 460-bed level I trauma center. PATIENTS: The study included 35 trauma/general surgery patients (16 in the open suction group, 19 in the closed suction group) who were treated with a total of 276 suctioning procedures (127 open, 149 closed). MEASUREMENTS AND MAIN RESULTS: Physiologic data collected after hyperoxygenation, immediately after suctioning, and 30 secs after suctioning, were compared with baseline values. Open endotracheal suctioning resulted in significant increases in mean arterial pressure throughout the suctioning procedure. Both methods resulted in increased mean heart rates. However, 30 secs after the procedure, the open-suction method was associated with a significantly higher mean heart rate than was the closed method. Closed suctioning was associated with significantly fewer dysrhythmias. Arterial oxygen saturation and systemic venous oxygen saturation decreased with open suctioning. In contrast, arterial oxygen saturation and systemic venous oxygen saturation increased with the closed suction method. There was no difference between the two methods in the occurrence of nosocomial pneumonia. Open endotracheal suctioning cost $1.88 more per patient per day and required more nursing time. CONCLUSIONS: The closed suction method resulted in significantly fewer physiologic disturbances. Closed suctioning appears to be an effective and cost-efficient method of endotracheal suctioning that is associated with fewer suction-induced complications.

Adult↗