Biomedical subjects

K L Meyer

Publications and source records attributed to K L Meyer.

At least 19 recordsLinked to original sources

PubMed2000

RNA-protein crosslinking to AMP residues at internal positions in RNA with a new photocrosslinking ATP analog.

A new photocrosslinking purine analog was synthesized and evaluated as a transcription substrate for Escherichia coli RNA polymerase. This analog, 8-[(4-azidophenacyl)thio]adenosine 5'-triphosphate (8-APAS-ATP) contains an aryl azide photocrosslinking group that is attached to the ATP base via a sulfur-linked arm on the 8 position of the purine ring. This position is not involved in the normal Watson-Crick base pairing needed for specific hybridization. Although 8-APAS-ATP could not replace ATP as a substrate for transcription initiation, once stable elongation complexes were formed, 8-APAS-AMP could be site-specifically incorporated into the RNA, and this transcript could be further elongated, placing the photoreactive analog at internal positions in the RNA. Irradiation of transcription elongation complexes in which the RNA contained the analog exclusively at the 3' end of an RNA 22mer, or a 23mer with the analog 1 nt from the 3' end, produced RNA crosslinks to the RNA polymerase subunits that form the RNA 3' end binding site (beta, beta'). Both 8-APAS-AMP and the related 8-azido-AMP were subjected to conformational modeling as nucleoside monophosphates and in DNA-RNA hybrids. Surprisingly, the lowest energy conformation for 8-APAS-AMP was found to be syn, while that of 8-azido-AMP was anti, suggesting that the conformational properties and transcription substrate properties of 8-azido-ATP should be re-evaluated. Although the azide and linker together are larger in 8-APAS-ATP than in 8-N(3)-ATP, the flexibility of the linker itself allows this analog to adopt several different energetically favorable conformations, making it a good substrate for the RNA polymerase.

Adenosine Monophosphate↗

PubMed2000

Nitric oxide interferes with islet cell zinc homeostasis.

Zinc is crucial for the biosynthesis, storage, and secretion of insulin in pancreatic islet cells. We have previously presented evidence that NO interferes with cellular Zn(2+) homeostasis and we therefore investigated the influence of chronic NO exposure on the labile islet cell Zn(2+) content. A strong fluorescence activity in a large islet cell subpopulation was found after staining with the Zn(2+)-specific fluorophore Zinquin. Culture for 24 h in the presence of nontoxic concentrations of the slow-releasing NO donor DETA/NO resulted in a significantly reduced Zn(2+)-dependent fluorescence. This appears to be islet specific as in endothelial cells DETA/NO exposure enhanced the Zn(2+)-dependent fluorescence activity in a concentration-dependent manner. These results suggest that NO interferes with cellular Zn(2+) homeostasis, which in islet cells is crucial for proper hormone delivery and thus special cell function.

Animals↗

PubMed2000

Enhanced green fluorescent protein fusion proteins of herpes simplex virus type 1 thymidine kinase and cytochrome P450 4B1: applications for prodrug-activating gene therapy.

To monitor therapeutic transgene expression, we developed fusion genes of enhanced green fluorescent protein (EGFP) with two different prodrug-activating enzyme genes: herpes simplex virus type 1 thymidine kinase (HSV-tk) and rabbit cytochrome P450 4B1 (cyp4b1). Expression of the resulting fusion proteins, TK-EGFP and 4B1-EGFP, rendered transduced human and rodent glioma cells sensitive to cytotoxic treatment with the corresponding prodrugs ganciclovir and 4-ipomeanol. Ganciclovir and 4-ipomeanol sensitivity was comparable with that achieved with the native HSV-TK and CYP4B1 proteins. As shown by fluorescence microscopy, TK-EGFP was expressed predominantly intranuclearly, whereas 4B1-EGFP was detectable in the cytoplasm, thereby displaying the orthotopic subcellular distribution of the corresponding native enzymes. The fluorescence intensity correlated well with the corresponding prodrug sensitivity, as shown by fluorescence-activated cell sorter analysis. EGFP expression was also used for the selection of stably HSV-tk-transduced cells by flow cytometric cell sorting. Resulting cell populations showed a homogeneity of fluorescence intensity similar to single-cell clones after antibiotic selection. In conclusion, tk-egfp and 4b1-egfp fusion genes are valuable tools for monitoring prodrug-activating gene therapy in living cells. EGFP fusion genes/proteins provide a simple and reproducible means for the detection, selection, and characterization of cells expressing enzyme genes for prodrug activation.

Animals↗

PubMed1999

Influence of nitric oxide on the intracellular reduced glutathione pool: different cellular capacities and strategies to encounter nitric oxide-mediated stress.

Different cell types exhibit huge differences towards the cytotoxic action of NO. In search for an explanation, we used subtoxic concentrations of the NO-donors S-nitrosocysteine (SNOC) for short-term challenge and of (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1- ium-1,2-diolate (DETA/NO) for longer periods of exposure, respectively, and subtoxic concentrations of the oxidant H2O2 to determine the impact on intracellular reduced glutathione (GSH) concentrations. We find that GSH concentrations are always decreased, but that different cell types show different responses. Incubation of the relatively NO-sensitive murine lymphocytes with both NO-donors, but not with H2O2, resulted in a nearly complete loss of intracellular GSH. Short-term NO-treatment of P815 mastocytoma cells, also sensitive to NO-mediated cell death, decreased GSH to a similar extent only if either glutathione reductase (GSHR) activity or y-glutamylcysteine synthetase (gammaGCS) activity were inhibited concomitantly by specific inhibitors. Long-term NO-treatment of P815 cells, however, resulted in a significant decrease of GSH that could be further enhanced by inhibiting gammaGCS activity. In contrast, neither short-term nor long-term NO-exposure nor H2O2-treatment affected intracellular GSH levels of L929 fibroblasts, which were previously shown to be extremely resistant towards NO, whereas concomitant gammaGCS inhibition, but not GSHR inhibition, completely decreased GSH concentrations. These results show that different cell types use different pathways trying to maintain glutathione concentrations to cope with nitrosative stress, and the overall capability to maintain a critical amount of GSH correlates with susceptibility to NO-induced cell death.

Animals↗

PubMed1999

Behavioral effects of psychomotor stimulant infusions into amygdaloid nuclei.

The role of amygdaloid nuclei in locomotion, stereotypy, and conditioned place preference (CPP) produced by psychomotor stimulants was examined. Five 2-day conditioning trials were conducted over 10 consecutive days. Rats received bilateral intracranial infusions of saline, cocaine (25-100 micrograms/side), or amphetamine (0.31-20 micrograms/side) into the ventricles (ICV), basolateral amygdala (BlA), or central amygdala (CeA) and were confined to a compartment. On alternating days, rats received sham infusions and were confined to a different compartment. Locomotion was measured daily, stereotypy was measured on trials 1 and 5, and CPP was measured 24 h after conditioning. ICV infusions of cocaine or amphetamine produced locomotion, rearing, and CPP. Intra-BlA and intra-CeA infusions of the highest dose of cocaine produced locomotion. In contrast, intra-CeA infusions of amphetamine potently produced locomotion and CPP. Intra-BlA infusions of amphetamine, however, did not produce any behavioral changes. These results suggest that the CeA, but not the BlA, is involved in initiating reward and locomotion produced by amphetamine.

Amphetamine↗

PubMed1997

Nitric oxide mediates intracytoplasmic and intranuclear zinc release.

We previously described that NO. leads to destruction of ZnS clusters and release of Zn2+ from various proteins including zinc finger transcription factors. To assess the relevance in living cells, we investigated, whether exogenous NO. leads to an increase of cytoplasmic and nuclear free Zn2+. L929 cells, mouse splenocytes, or rat aorta endothelial cells were labeled with Zinquin-E, a Zn2+-specific fluorophore, and were treated with two different spontaneous NO donors, S-nitrosocysteine or DETA/NO. Both NO donors strongly increased the Zn2+-dependent fluorescence in the cellular cytosol and also in nuclei as compared to controls. NO-dependent Zn2+ release in splenocytes was quantitated by flow cytometry. These results show for the first time, that nitrosative stress mediates intracellular and intranuclear Zn2+ release which may be relevant in altering gene expression patterns.

Animals↗

PubMed1996

Sensitivity-enhanced echo-planar MRI at 1.5T using a 5 x 5 mesh dome resonator.

In this work a 5 x 5 mesh dome resonator that has been optimized for functional brain imaging is presented. The resonator was reduced in length and diameter compared with previous versions to reduce sample losses, thus enhancing the signal-to-noise ratio of the acquired data. In addition, a 5 x 5 mesh design was employed, which offered improved axial homogeneity over an earlier 3 x 3 mesh version. The new resonator exhibited high sensitivity and good homogeneity over the brain volume, permitting analysis of functional activation over large areas of the cerebral cortex. In a direct comparison with a standard clinical head-imaging resonator, the high sensitivity of the 5 x 5 mesh dome resonator resulted in greater statistical confidence in functional activation.

Cerebral Cortex↗

PubMed1996

Effect of 6-aminonicotinamide on the pentose phosphate pathway: 31P NMR and tumor growth delay studies.

6-aminonicotinamide (6AN) has been shown to enhance radiosensitivity in vitro, although previous in vivo studies failed to show an effect. 31P NMR spectra were obtained by using a one-dimensional chemical shift imaging technique on a first generation transplant of the CD8FI spontaneous mammary carcinoma tumor model. Spectra were obtained both before and 10 h after treatment with 6AN (20 mg/kg). Changes in pH, nucleoside triphosphate/inorganic phosphate, and phosphocreatine/ inorganic phosphate measured at 10 h post-6AN were not significant. A new peak was detected 10 h post-6AN, which was assigned to 6-phosphogluconate (6PG), indicating inhibition of the pentose phosphate pathway (PPP). Based on the spectral data demonstrating inhibition of the PPP at 10 h post-6AN, tumor-bearing mice were irradiated (15 Gy x 3 fractions) on Days 1, 10 or 11, and 21 10 h after administration of 6-aminonicotinamide (20 mg/kg). Tumor-bearing mice receiving 6AN alone (20 mg/kg x 3), radiation alone (15 Gy x 3), or saline were also studied. Tumor growth delay studies indicated that 6AN alone induced a small but significant tumor growth delay (4.3 +/- 0.8 days). Radiation alone induced a tumor growth delay of 34.5 +/- 2.7 days. Treatment with 6AN followed by radiation induced a tumor growth delay of 57.0 +/- 3.8 days. This was significantly greater than the TGD values for treatment with 6AN alone or radiation (P < 0.01). No complete regressions were noted after treatment with 6AN or radiation alone. Concomitant therapy with 6AN plus radiation yielded 6/28 complete regressions (21%), which was significantly greater than radiation (P < 0.05) or 6AN alone (P < 0.01) on this mammary carcinoma.

6-Aminonicotinamide↗

PubMed1995

Nitric oxide induces apoptosis in mouse thymocytes.

Nitric oxide (NO) produced at high concentrations by the inducible NO synthase is an important effector molecule involved in immune regulation and defense. We have examined whether NO represents a signal for triggering apoptosis in thymocytes. Freshly isolated thymocytes were incubated with different chemical NO donors for various intervals. Apoptosis was determined by detection of DNA strand breaks with in situ nick translation. All NO donors induced thymocyte apoptosis with 30% positive thymocytes vs 10% in controls after 8 h. Apoptosis was prevented by addition of ZnSO4. Short-term pre-exposure to NO resulted in protection from apoptosis induced by glucocorticoids comparable with the protective effect of heat shock. Flow cytometry revealed that NO treatment as well as heat shock or dexamethasone incubation is accompanied by reduction in the CD4+ CD8+ thymocyte subpopulation. Apoptosis induction was accompanied by increased expression of p53, as detected by PCR analysis 2 h after NO donor addition. In vivo treatment of mice with endotoxin results in increased thymic apoptosis. Focal apoptosis was found to occur in close proximity to blood vessels 18 h after LPS treatment. Capillary endothelium and dendritic cells adjacent to apoptotic foci were found to stain strongly for inducible NO synthase expression. Furthermore, in an in vitro experiment using cocultures of thymocytes with LPS/cytokine-activated endothelial cells expressing inducible NO synthase, a significantly increased rate of thymocyte apoptosis was found, and this could be prevented completely by inhibiting NO production. Addition of dexamethasone to these cocultures did not lead to a further increase in the percentage of apoptotic thymocytes, underlining the protective effect of NO on dexamethasone-induced apoptosis.

Animals↗

PubMed1995

A 3 x 3 mesh two-dimensional ladder network resonator for MRI of the human head.

A new radiofrequency resonator for MR imaging of the human head is presented. The geometry consists of a single end-ring connected to eight legs which extend along a cylinder and are joined in pairs on a hemispherical dome. The most homogeneous normal mode of this structure is doubly degenerate and affords quadrature operation. The high sensitivity in the hemispherical end is particularly suited to human brain studies. This resonator represents a clinical application of two-dimensional ladder network resonant structures whose operation may be understood by analogy to the mechanical problem of oscillating two-dimensional membranes.

Adult↗

PubMed1994

Molecular structures and conformational studies of triarylcyclopropyl and related nonsteroidal antiestrogens.

Molecular structures and conformational characteristics of a series of 1,1-dichloro-2,2,3-triarylcyclopropanes (DTACs), which were reported previously to be distinctly antiestrogenic and inhibitors of the estrogen-receptor-positive MCF-7 human breast cancer cells in culture, are reported. In addition, structural and conformational features of the DTACs were compared to the first-known nonsteroidal antiestrogen, MER25, and the clinically useful antiestrogen Tamoxifen. The molecular structures of four DTAC compounds were determined by X-ray diffraction. Crystallographic structures show that the DTAC molecules have nearly the same relative conformation for the three aryl rings which is designated as a "nonpropeller" conformation in contrast to the observed "propeller" conformation for the three rings in all known triarylethylenes. Systematic conformational searches were performed to find the conformational preferences of DTACs, MER25, and Tamoxifen using idealized model compounds built from their respective crystal structure. Energy-minimization and conformational-search studies demonstrated that all DTAC molecules have a common, single global minimum energy conformer for their central core containing the dichlorotriarylcyclopropyl system, which is similar to that found in their crystal structures. Conformational search of MER25 showed that the molecule can assume a number of low-energy conformers of which two, one anti (A1) and one gauche (G1A), have about the same energy. The anti conformation is similar to the one observed in its crystal structure and resembles the estrogenic E-isomer of Tamoxifen, while the lowest energy gauche conformer of MER25 resembles more closely the antiestrogenic Z-isomer of Tamoxifen. NMR spectroscopic analysis of MER25 showed that the molecule exists predominantly in the anti conformation in solution. A comparative review of the structural features and bioactivities of Tamoxifen, DTACs, and MER25 provides a possible explanation for their low estrogen receptor binding affinity which is common to these compounds together with their antiestrogenic activity.

Crystallization↗

PubMed1994

(Z)-1,1-dichloro-2-(4-benzyloxyphenyl)-2,3-bis(4-methoxyphenyl)cyclopropane: the synthesis and enantiomeric separation of an antitumor agent.

(Z)-1,1-dichloro-2-(4-benzyloxyphenyl)-2,3-bis(4-methoxyphenyl)cyc lopropane (5), a potential antitumor agent designed to treat breast cancer, was prepared in three steps. A stereospecific palladium-catalyzed cross coupling reaction which provided the intermediate (Z)-triaryl alkene 4 was a crucial step in the synthesis. Makosza phase transfer reaction on 4 gave the enantiomeric (Z)-dichlorocyclopropane derivatives 5 which were resolved by semipreparative HPLC on a chiral stationary phase consisting of amylose tris-3,5-dimethylphenyl carbamate coated on silica gel.

Antineoplastic Agents↗

PubMed1994

Demonstration of differences in vascular permeability in experimental tumors by use of 19F magnetic resonance imaging.

RATIONALE AND OBJECTIVES: In vivo assessment of tumor vascular permeability may provide useful information for chemotherapy treatment planning or for the assessment of treatment effectiveness. We aimed to assess vascular permeability in two tumor sublines as well as changes in vascular permeability with tumor growth by using 19F magnetic resonance imaging. METHODS: An emulsion of perfluorotributylamine was used as a tumor extravascular contrast agent for 19F MRI. The amount of emulsion that leaked into tumor interstitial space was analyzed qualitatively with imaging. A quantitative study of vascular permeability was done with a separate group of tumors by use of Evans blue dye. RESULTS: One tumor type was more permeable to both perfluorotributylamine emulsion and Evans blue than was the second tumor type. The difference was attributed to a difference in surface area for exchange. In larger tumors of both types, pooling of large amounts of perfluorocarbon occurred and was assumed to be attributable to hemorrhage or blood flow stasis or both. CONCLUSION: 19F MRI is capable of demonstrating the permeability of tumor vessels to macromolecular substances.

Adenocarcinoma↗

PubMed1993

Synthesis of phosphocholine and quaternary amine ether lipids and evaluation of in vitro antineoplastic activity.

The in vitro antineoplastic activity of many phosphorus-containing (e.g., phosphocholines) and non-phosphorus-containing (e.g., quaternary ammonium salts) ether lipids has been evaluated in the HL-60 promyelocytic cell line. These compounds are analogues of ET-18-OMe (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine). Structural modification of 1-(alkylamido)-, -(alkylthio)-, and -(alkyloxy)propyl backbones has provided further insight into the structure-activity relationships of these lipids. In this study, a long saturated C-1 chain and a three-carbon backbone with a single short C-2 substituent were preferred. At the positively charged nitrogen of phosphocholines, fewer than three substituents caused a significant loss of activity, and substituents larger than methyl decreased activity slightly. In the nonphosphorus compounds, many nitrogen heterocycles and also a sulfonium moiety were incorporated without changing the degree of activity; however, a thiazolium group decreased activity. The most active compound, 29 [N-[3-(hexadecyloxy)-2-methoxypropyl]-3-(hydroxymethyl)pyridinium bromide], was approximately twice as active as the reference standard, ET-18-OMe, in a trypan blue dye exclusion assay.

Antineoplastic Agents↗

PubMed1993

The Acute Physiology and Chronic Health Evaluation II classification system is a valid marker for physiologic stress in the critically ill patient.

OBJECTIVE: To compare the Acute Physiology and Chronic Health Evaluation (APACHE II) score with resting energy expenditure obtained from indirect calorimetry to determine whether the APACHE II scoring system is an accurate, objective measure of the degree of critical illness and physiologic stress between groups of patients. DESIGN: Prospective study. SETTING: University hospital, tertiary referral center. PATIENTS: Seventy critically ill patients, consecutively sampled from burn, surgical, and medical intensive care units. INTERVENTIONS: Indirect calorimetric studies were performed on each patient using a metabolic cart. The acute physiologic score component of the APACHE II scoring system was determined at the time of metabolic testing, a mean of 15.9 days after hospital admission. MEASUREMENTS AND MAIN RESULTS: True resting energy expenditure was calculated by adjusting the measured energy expenditure for diet-induced thermogenesis and fever. A predicted resting energy expenditure was calculated for each patient using the Harris-Benedict equation alone, and by using the Harris-Benedict value corrected for previously published metabolic activity factors. To eliminate differences in body composition and size, true resting energy expenditure was divided by weight, body surface area, and Harris-Benedict resting energy expenditure. Results showed no significant correlation between APACHE II scores and either the Harris-Benedict resting energy expenditure or the Harris-Benedict value corrected by metabolic activity factors. However, there was a significant (p < or = .001; r2 = .18 to .20) relationship between increasing APACHE II scores and both increasing measured and true resting energy expenditure. The true resting energy expenditure divided by body surface area, kilogram body weight, and Harris-Benedict predicted value, were all shown to be significantly (p < .01) related to APACHE II score, but showed no better degree of correlation (r2 = .12 to .23) than comparison of APACHE II score with measured or true resting energy expenditure. CONCLUSIONS: The APACHE II classification may be a valid marker of physiologic stress as demonstrated by its statistically significant (although weak) relationship with indirect calorimetric measures of energy expenditure associated with varying degrees of critical illness.

Adolescent↗

PubMed1993

Measurement of vascular volume in experimental rat tumors by 19F magnetic resonance imaging.

RATIONALE AND OBJECTIVES: In-vivo assessment of tumor vascular volume may provide useful information for treatment planning or the assessment of treatment effectiveness. The goals of our study were to measure percent vascular volume in two experimental tumor sublines using 19F magnetic resonance imaging (MRI) and to assess changes in tumor blood volume with growth. METHODS: An emulsion of perfluorotributylamine (FTBA) was used as a vascular contrast agent for 19F MRI: The amount of emulsion in the tumor vasculature was measured by 19F MRI and used to calculate percent vascular volume. A separate ex-vivo study of vascular volume was conducted using the dye Hoechst 33342. A total of five rats were studied by MRI and 14 by the ex-vivo method. RESULTS: The ranges of percent vascular volume values measured in the imaging and Hoechst dye studies were 2% to 9% and 1.25 to 7%, respectively. A trend toward decreasing percent vascular volume with increasing tumor volume was evident in one tumor subline. CONCLUSIONS: The quantitative 19F MRI technique was effective for measuring percent vascular volume and changes in vascular volume with growth.

Adenocarcinoma↗

PubMed1992

Fluorinated blood substitute retention in the rat measured by fluorine-19 magnetic resonance imaging.

RATIONALE AND OBJECTIVES: Emulsions of perfluorocarbons (PFCs) have been tested as blood substitutes. However, evidence exists that there is long-term retention of some PFCs by the organs of the reticuloendothelial system (RES). The authors investigate organ retention of the blood substitute component, perfluorotripropylamine (FTPA), using fluorine-19 (19F) magnetic resonance imaging (MRI). METHODS: Various dosages of an emulsion of FTPA were administered to five rats. At intervals up to 86 weeks after infusion, 19F MRI was used to measure the amount of FTPA in liver and spleen. The data were fit to both linear and exponential elimination models, and organ retention half-lives were calculated. RESULTS: The exponential half-lives for combined liver and spleen FTPA ranged from 110 to 190 days. Linear half-lives ranged from 175 to 300 days. CONCLUSIONS: FTPA retained by the liver and spleen may be quantified by 19F MRI: The half-lives that were measured are longer than those reported previously for FTPA.

Animals↗

PubMed1991

Expansion of a maternally derived monoclonal T cell population with CD3+/CD8+/T cell receptor-gamma/delta+ phenotype in a child with severe combined immunodeficiency.

The diagnosis of severe combined immunodeficiency complicated by chronic graft-vs-host disease affecting liver and skin in association with engraftment of maternal T cells was established in a 5-mo-old boy. Detailed immunologic and molecular genetic studies were performed because a unique T cell phenotype was identified on initial evaluation. A major proportion of the patient's peripheral T cells expressed a CD8+ and TCR-gamma/delta+ phenotype while CD4+ T cells were virtually absent. Southern blot analysis of cell subpopulations isolated by fluorescence activated cell sorting indicated that approximately 50% of CD8+/TCR-gamma/delta+ cells were clonally related. Immunophenotyping and -genotyping also identified a clonal TCR-gamma/delta+ cell population in the child's mother. Clonal identity of these T cell populations in mother and child was demonstrated by studies using a clonspecific TCR-delta probe generated by polymerase chain reaction as well DNA sequence analysis. HLA typing and DNA fingerprinting confirmed that the child had acquired this clone diaplacentally from the mother. According to immunohistology and DNA analysis the clone was found to be virtually absent in the liver tissue suggesting that this clonal T cell population plays a minor role, if any, in the pathogenesis of the liver abnormalities in the patient. In the mother the CD8+/TCR-gamma/delta+ clone spontaneously declined to a level around 1% of PBMC several months later and has remained at this level since. We conclude that 1) a clonal expansion of TCR-gamma/delta T cells, triggered by yet unknown stimuli, may occur in otherwise healthy individuals, 2) respective T cells are able to cross the placental barrier, and 3) in an microenvironment precluding rejection, i.e., in severely immunocompromised patients, these cells may persist and even represent a significant proportion of circulating T cells.

Alleles↗