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Biomedical subjects

D Zacharias

Publications and source records attributed to D Zacharias.

16 recordsLinked to original sources
PubMed2001

Fluorescence resonance energy transfer analysis of cell surface receptor interactions and signaling using spectral variants of the green fluorescent protein.

BACKGROUND: Fluorescence resonance energy transfer (FRET) is a powerful technique for measuring molecular interactions at Angstrom distances. We present a new method for FRET that utilizes the unique spectral properties of variants of the green fluorescent protein (GFP) for large-scale analysis by flow cytometry. METHODS: The proteins of interest are fused in frame separately to the cyan fluorescent protein (CFP) or the yellow fluorescent protein (YFP). FRET between these differentially tagged fusion proteins is analyzed using a dual-laser FACSVantage cytometer. RESULTS: We show that homotypic interactions between individual receptor chains of tumor necrosis factor receptor (TNFR) family members can be detected as FRET from CFP-tagged receptor chains to YFP-tagged receptor chains. Noncovalent molecular complexation can be detected as FRET between fusions of CFP and YFP to either the intracellular or extracellular regions of the receptor chains. The specificity of the assay is demonstrated by the absence of FRET between heterologous receptor pairs that do not biochemically associate with each other. Interaction between a TNFR-like receptor (Fas/CD95/Apo-1) and a downstream cytoplasmic signaling component (FADD) can also be demonstrated by flow cytometric FRET analysis. CONCLUSIONS: The utility of spectral variants of GFP in flow cytometric FRET analysis of membrane receptors is demonstrated. This method of analyzing FRET allows probing of noncovalent molecular interactions that involve both the intracellular and extracellular regions of membrane proteins as well as proteins within the cells. Unlike biochemical methods, FRET allows the quantitative determination of noncovalent molecular associations at Angstrom level in living cells. Moreover, flow cytometry allows quantitative analyses to be carried out on a cell-by-cell basis on large number of cells. Published 2001 Wiley-Liss, Inc.

Adaptor Proteins, Signal Transducing↗
PubMed2001

2-Azabicyclo[2.1.1]hexanes. 2. Substitutent effects on the bromine-mediated rearrangement of 2-azabicyclo[2.2.0]hex-5-enes.

Methyl- and phenyl-substituted N-(ethoxycarbonyl)-2-azabicyclo[2.2.0]hex-5-enes 6 have been prepared by photoirradiation of appropriately substituted 1,2-dihydropyridines. Torquoselectivity is observed in the synthesis of the 3-endo-methyl- and 3-endo-phenyl-2-azabicyclo[2.2.0]hexenes 6c-e from 2-methyl- and 2-phenyl-1,2-dihydropyridines 5c-e. Products formed upon addition of bromine to 3-endo-, 4-, and 5-methyl- and 3-endo-phenyl-substituted N-(ethoxycarbonyl)-2-azabicyclo[2.2.0]hex-5-enes 6a-f were substituent dependent. For 6a,b, which lack substituents at C(3) or C(5), mixtures of unrearranged dibromides 8a,b and rearranged dibromides 9a,b were obtained. With the 3-endo-substituents in 6c-e, only rearranged dibromides 9c-e were formed; 5-methyl substitution afforded mainly unrearranged dibromide 8f and some allylic bromide 10. Both unrearranged 5-endo,6-exo-dibromo-2-azabicyclo[2.2.0]hexanes 8 and rearranged 5-anti-6-anti-dibromo-2-azabicyclo[2.1.1]hexanes 9 are formed stereoselectively. The dibromoazabicyclo[2.1.1]hexanes 9 have been reductively debrominated to afford the first reported 2-azabicyclo[2.1.1]hexanes 11 with alkyl or aryl substituents at C-3.

Bridged Bicyclo Compounds, Heterocyclic↗
PubMed2000

Filgrastim as an alternative to donor leukocyte infusion for relapse after allogeneic stem-cell transplantation.

PURPOSE: Donor leukocyte infusion (DLI) effectively treats relapse after allogeneic stem-cell transplantation (alloSCT), but the response may require several months and may be associated with significant toxicity. Filgrastim has also been observed to effectively treat leukemic relapse after alloSCT. A retrospective analysis was performed to determine the effectiveness of filgrastim in treating relapses after alloSCT. PATIENTS AND METHODS: Fourteen patients with hematologic malignancies were treated with filgrastim at relapse after alloSCT. Filgrastim was given at 5 mcg/kg/d subcutaneously for 21 consecutive days. Response was evaluated at 7 days after completion of filgrastim. Immunosuppressants, if present, were rapidly tapered to complete discontinuation at the time of relapse. RESULTS: Three patients were not assessable for response because additional therapy was necessary before completion of filgrastim. Six patients (43%) achieved a complete response on an intent-to-treat basis. When response was evaluated based on relapse type, three of four cytogenetic relapses, two of three morphologic relapses, and one of four hematologic relapses achieved a complete remission. Two responses were observed in patients who were completely off of any immunosuppression at the time of relapse. Six patients developed chronic graft-versus-host disease. The event-free and overall survival rates for all 14 patients are 43% and 73%, respectively. CONCLUSION: The use of filgrastim with rapid discontinuation of immunosuppression results in response rates that are similar to results using DLI. Filgrastim could be considered as an alternative or an adjunct to DLI for relapses after alloSCT.

Adult↗
PubMed1997

Allogeneic-blood stem-cell collection following mobilization with low-dose granulocyte colony-stimulating factor.

PURPOSE: The optimal dose of granulocyte colony-stimulating factor (G-CSF) for mobilization of allogeneic-blood stem cells (AlloBSC) has yet to be determined. As part of a prospective trial, 41 related human leukocyte antigen (HLA)-matched donors had blood cells mobilized with G-CSF at 5 micrograms/kg/d by subcutaneous administration. The purpose of this trial was to monitor adverse effects during G-CSF administration and stem-cell collection, to determine the optimal timing for stem-cell collection, and to determine the cellular composition of stem-cell products following G-CSF administration. PATIENTS AND METHODS: The median donor age was 42 years. Apheresis began on day 4 of G-CSF administration. At least three daily 12-L apheresis collections were performed on each donor. A minimum of 1.0 x 10(6) CD34+ cells/kg (recipient weight) and 8.0 x 10(8) mononuclear cells/kg were collected from each donor. All collections were cryopreserved in 5% dimethyl sulfoxide and 6% hydroxyethyl starch. RESULTS: Toxicities associated with G-CSF administration and the apheresis process included myalgias/arthralgias (83%), headache (44%), fever (27%), and chills (22%). The median baseline platelet count of 242 x 10(4)/ mL decreased to 221, 155, and 119 x 10(6)/mL on days 4, 5, and 6 of G-CSF administration, respectively. Median numbers of CD34+ cells in collections 1, 2, and 3 were 1.99, 2.52, and 3.13 x 10(6)/kg, respectively. The percentage and total number of CD4+, CD8+, and CD56+/CD3- cells remained relatively constant during the three collections. Median total numbers of cells were as follows: CD34+, 7.73 x 10(6)/kg; and lymphocytes, 6.93 x 10(8)/kg. CONCLUSION: Relatively low doses of G-CSF can mobilize sufficient numbers of AlloBSC safely and efficiently.

Adult↗
PubMed1996

Buyouts & ventures. Selling ... or selling out.

Charity begins at home, yet across the country hometown not-for-profit hospitals are forming joint partnerships with investor-owned systems. In some of these cases no foundation is created to continue the hospital's good deeds. In this special report, Hospitals & Health Networks looks at the issues and asks, "Do these joint ventures need more public scrutiny?"

Charities↗
PubMed1993

Developmental expression of TERM-1 glycoprotein on growth cones and terminal arbors of individual identified neurons in the grasshopper.

The chemoaffinity theory postulates the existence of cell-specific molecular signals that uniquely identify individual developing neurons. Such molecules are thought to promote both accurate axon outgrowth and the formation of correct synaptic connections. To identify candidates for such neuron-specific recognition molecules, we generated monoclonal antibodies that recognize surface-associated antigens expressed by individual identified neurons in the grasshopper embryo. Here we report on a molecular label that is expressed exclusively by two pairs of sibling interneurons in the developing CNS. Our experiments indicate that during axogenesis, this molecule is expressed at the surface of the growth cones of these cells, while during subsequent synaptogenesis, it becomes concentrated at the cells' developing terminal arbors. In both cases the molecule appears to be secreted by the labeled structures. This molecule, which we call TERM-1, is a glycoprotein with a molecular weight of approximately 48 kDa. The highly restricted spatiotemporal expression pattern of TERM-1 implies that individual developing neurons can acquire and retain unique molecular labels that may be important for neuron-specific outgrowth and target recognition.

Animals↗
PubMed1991

Attractiveness of the male Acheta domesticus calling song to females. III. The relation of age-correlated changes in syllable period recognition and phonotactic threshold to juvenile hormone III biosynthesis.

1. Most crickets first demonstrated positive phonotaxis to 65 dB CSs having a 53-62 ms SP by day 3 following the imaginal molt (Fig. 3B). The onset of copulatory readiness occurred on average at 3.2 days. 2. The attractive range of SPs for most females became progressively broader as they aged (Fig. 4). Three to 4-day-old females were attracted to a smaller number of CS SPs than were 20-21 day old females (Fig. 4). 3. Older, less selective females did not typically respond to the same range of CS SPs (Fig. 6). However, they were more likely to respond to some SPs (especially 50 ms) than to others (Fig. 7). 4. The phonotactic threshold decreased from 95 dB or greater on day 0 to a mean of 55 dB by day 3, during a period of increasing JHIII biosynthesis, and thereafter remained at that level (Fig. 8). 5. During a period of maximal JHIII production, 3-5 day-old females usually responded to 4 of the 7 SPs presented (Fig. 8). Females older than 12 days were unselective for CS SP, and JHIII synthesis remained at a level below the peak production on day 4 (Fig. 8). 6. Older females, that were unselective for CS SP, became as selective as 3 to 5-day-old females within 4 days of topical application of JHIII (Figs. 9-11).

Aging↗
PubMed1991

Regulation of cricket phonotaxis through hormonal control of the threshold of an identified auditory neuron.

1. The phonotactic threshold of 3 to 5-day-old adult female Acheta domesticus and the threshold of the L1 auditory neuron drop progressively (Fig. 1). 2. Application of juvenile hormone III (JHIII) to 1-day-old females caused both the female's threshold for phonotaxis and the threshold of the L1 auditory neuron to drop 20 or more dB over the next 12 h (Figs. 3-4). 3. JHIII's effect on phonotactic threshold could be blocked by injection with a transcription (alpha-amanitin) or a translation blocker (emetine, Fig. 3). 4. Injection of emetine also prevented the JHIII induced drop in L1's threshold (Fig. 4). 5. Application of JHIII to the surface of, or microinjection of JHIII into one prothoracic hemiganglion caused the female to circle phonotactically away from the side of hormone addition at thresholds 25 to 35 dB lower than the pre-JHIII addition threshold within 2 h (Fig. 6). 6. Application of JHIII to the surface of both prothoracic hemiganglia, accompanied by microinjection of emetine into one hemiganglion resulted in the female emetine into one hemiganglion resulted in the female circling phonotactically toward the side receiving emetine injection, with a 25 to 35 dB drop in threshold (Fig. 6).

Animals↗
PubMed1991

A prospective evaluation of glucose reagent teststrips in the prehospital setting.

Reagent teststrip determination of blood glucose has been shown to be accurate for hospital and home testing and is commonly used in prehospital care despite the lack of studies in this arena. This prospective, multicenter study examines the ability of glucose reagent teststrips to detect hypoglycemia when used under field conditions compared with simultaneously drawn control samples for laboratory glucose determination. Also examined was the accuracy of the teststrips in the laboratory glucose range less than or equal to 200 mg/dL. One hundred eighty-one pairs of data were analyzed. Hypoglycemia was defined as laboratory glucose less than or equal to 60 mg/dL. The teststrips correctly identified 31 of 33 patients in this range (sensitivity = 94%), and 125 of 148 patients without hypoglycemia (specificity = 85%). The two false negative readings were 70 and 90 mg/dL. Reagent teststrips were within +/- 40 mg/dL of the laboratory value in 70% of cases. The correlation coefficient (Spearman r) between teststrip and laboratory glucose in the range less than or equal to 200 mg/dL was .80. Using teststrip readings of 90 mg/dL or less as a measure of hypoglycemia yields 100% sensitivity with a specificity of 57%. We conclude glucose reagent teststrips are a useful adjunct for use in the prehospital setting and may be valuable for the detection of hypoglycemia.

Analysis of Variance↗
PubMed1989

Correlation of nonlinear mathematical transformation of the normal electrocardiogram with the severity of coronary artery disease.

Signal processing was applied to normal ECGs to study whether the results could predict coronary artery disease (CAD) on angiography. ECGs determined to be normal by conventional criteria for 116 white males and 84 white females were subjected to signal processing. The technique applied nonlinear mathematical transformation to a segment of seven ECG leads which were photographically imaged, automatically digitized, normalized for time, and had topographical coordinate transformation using an IBM-XT microcomputer. The resulting curvilinear display on the computer monitor was termed a biopotential coordinate transformation (BCT). Successive superimposition of BCTs from normal ECGs of patients with normal angiograms developed boundaries of a normal template for each lead. Study BCTs were then compared to the normal templates to predict the presence or absence of CAD. Correlation of the BCT results with the severity of CAD for the males resulted in average sensitivities of 80%, 84.4%, and 91.7% for single, double, and triple-vessel CAD, respectively. Similarly, for the females average sensitivities were 59.1%, 73.9%, and 88.9%, respectively. In past studies on the same population, the BCT process detected CAD with average sensitivity of 85.1% and specificity of 81.8% for white males and average sensitivity of 73% and specificity 81% for white females. As such, computerized signal processing of ECGs may provide a noninvasive method for detection of CAD in patients with seemingly normal resting ECGs. The results of this study indicate that the sensitivity of the process increases in direct proportion to the severity of CAD. Further investigation is warranted for process refinement and verification.

Coronary Angiography↗
PubMed1988

Detection of coronary artery disease from the normal resting ECG using nonlinear mathematical transformation.

In order to enhance subtle changes not apparent in resting ECGs by conventional criteria, a computerized signal processing technique, biopotential coordinate transformation (BCT), has been developed to increase the physician's perceptibility of CAD in seemingly "normal" resting ECGs. ECGs initially read as normal then can be tested with this process to predict the presence or absence of CAD. A blinded, retrospective study of 93 Caucasian patients with normal ECGs was performed using coronary angiography as the test standard. The BCT process identified the presence or absence of CAD with an 84.3% sensitivity and 81.8% specificity in 62 men. Similarly, a 76.2% sensitivity and 80% specificity was obtained for 31 women. These results suggest that the BCT process may provide the emergency physician with a noninvasive screening test for the detection of CAD in patients with apparently normal resting ECGs. Further studies are necessary for process refinement and verification.

Coronary Angiography↗
PubMed1986

Diagnosis of complex acid-base disorders: physician performance versus the microcomputer.

Patients with acid-base disturbances that are often complex frequently present to the emergency department. The sometimes hectic nature of the ED can preclude the appropriate quantitative analysis required by these disorders, especially when mixed disturbances are present. A computer program using generally accepted acid-base and electrolyte formulae was developed for use on the Apple II+ or IBM-PC microcomputer. Each of a series of 35 acid-base disturbances incorporating single, double, and triple disorders was correctly identified by the computer in less than 45 seconds. Problem sets based on the same 35 disturbances were presented to 21 physician-subjects at various levels of training from the emergency medicine, internal medicine, pediatrics, surgery, and family practice specialties. Although the physicians were given unlimited time and the necessary formulae to reach a diagnosis, they were requested to perform their analyses in the same fashion used in the ED. Although times varied widely, no physician spent more than five minutes on any problem. The physician correct response rates were 86%, 49%, and 17% for single, double, and triple disorders, respectively. The primary disorder correct response rate was 89% for double disorders and 94% for triple disorders. The primary and secondary disorder correct response rate was 58% for triple disorders. The data suggest that the microcomputer may be beneficial in the rapid assessment of complex disorders.

Acid-Base Imbalance↗
PubMed1986

Nonlinear transformation of the resting electrocardiogram in the diagnosis of coronary artery disease.

Despite the common use of the standard 12-lead ECG, its reliability as an indicator of coronary artery disease (CAD) is poor. The normal ECG is falsely negative in more than 50% of angiographically proven CAD. The waveform of the standard ECG, however, can be transformed mathematically by nonlinear signal transformation to enhance its interpretation by computer. Using such a process it is possible that abnormalities can be identified in "normal" ECGs that can be correlated with CAD, thus identifying high-risk patients. A computer template that represents grouped data of normal ECGs for patients who also have normal coronary angiography was developed. Unblinded, preliminary testing of the template on normal ECGs of 107 white patients who had normal or abnormal coronary angiograms was performed. The process identified presence or absence of CAD with 82% specificity and 71% sensitivity for 53 women, and with 82% specificity and 86% sensitivity for 54 men. These preliminary results are promising, but further refinement of the templates is required and blinded studies with larger numbers and varieties of patients are needed.

Angiography↗
PubMed1986

A comparative histopathological, immunological, and biochemical study of experimental intravenous paracoccidioidomycosis induced in mice by three Paracoccidioides brasiliensis isolates.

We carried out a comparative study of the histopathology (lung, liver, spleen, kidney and adrenals) and the anti-P. brasiliensis humoral (immunodiffusion test) and cellular (footpad test) immune response of mice intravenously inoculated with yeast forms of three P. brasiliensis isolates (Pb 18, Pb 192, Pb 265). Pb 265 (avirulent strain) did not evoke specific lesions or antibody production; the levels of cellular immunity were significantly lower than those of the two other isolates. Lung granulomas induced by strain Pb 18 were richer in fungi and neutrophils and poorer in mononuclear cells when compared to those induced by strain Pb 192. Extrapulmonary lesions were more frequent in mice infected with strain Pb 18. Strains Pb 18 and Pb 192 raised similar humoral and cellular anti-P. brasiliensis responses. Cell wall analysis did not suggest striking differences among the strains. Slightly higher levels of the water soluble fraction 3 (which contains the immunogenic galactomannan and protein) were detected in strain Pb 265.

Animals↗