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L Evangelista

Publications and source records attributed to L Evangelista.

9 recordsLinked to original sources
PubMed1999

A homogeneous and multiplexed immunoassay for high-throughput screening using fluorometric microvolume assay technology.

We have developed a simple, homogeneous bead-based immunoassay for use with fluorometric microvolume assay technology (FMAT). The FLISA (fluorescence-linked immunosorbent assay) can be easily adapted from existing immunoassays, is comparable to traditional ELISAs with respect to linear dynamic range and sensitivity, and can be readily performed in 96- and 384-well plates. Additionally, the FLISA utilizes 100-fold less primary antibody than the conventional immunoassay. The scanner uses a helium/neon laser to image and measure bead-bound fluorescence while the background fluorescence is ignored. Consequently, no wash steps are required to remove unbound antibody, ligand, and fluorophore. Furthermore, the instrument is capable of detecting two different fluorescent dyes, allowing for multiplexed assays based on color. Fluorescent bead-based immunoassays were developed for the cytokines IL-6 and IL-8, and their use in both one-color and two-color FLISAs is demonstrated. Although no wash steps were employed, the FLISA was able to accurately measure the concentrations of IL-6 and IL-8 in the growth media of cytokine-stimulated HUVEC cells. In addition, a simulated high-throughput two-color FLISA positively identified those wells in a 384-well plate that contained different amounts of IL-6 and/or IL-8 peptide. The homogeneous, multiplex and multiplate format of the FLISA reduces hands-on time and reagent usage, and is therefore ideally suited for high-throughput screening.

Cells, Cultured↗
PubMed1999

Biological activity of Melaleuca alternifola (Tea Tree) oil component, terpinen-4-ol, in human myelocytic cell line HL-60.

BACKGROUND: Tea tree oil is an aboriginal Australian traditional medicine for bruises, insect bites, and skin infections. It was rediscovered in the 1920s as a topical antiseptic that is more effective than Phenol. Previous studies have demonstrated its antiseptic qualities, but its effects on human white blood cells have never been investigated. OBJECTIVE: To test the hypothesis that tea tree oil exerts its antiseptic action through white blood cell activation. METHODS: Crude oil and the purified "active" component were studied by using a model system that responds to bioactive components by induction of differentiation in white blood cells. Methods used included white blood cell oxidative burst assay (nitroblue tetrazolium [NBT] dye reduction); cell proliferation assay (tritiated thymidine incorporation); cell surface differentiation marker assay (flow cytometric quantitation of phycoerythrin-anti-CD 11b binding); cell viability assay (trypan blue exclusion); and cellular differentiation enzyme assay (white cell esterase staining). RESULTS: Collectively, five assays that measure differentiation in white blood cells indicated monocytic differentiation after treatment with either crude oil or the purified active component. Both the crude oil and the purified active component, (+:-) terpinene-4-ol, caused a similar type and amount of differentiation. The culture of cells in medium containing serum caused more activation than in medium containing no serum. CONCLUSION: The antiseptic activity of tea tree oil appears to be due, in part, to white blood cell activation.

Anti-Infective Agents, Local↗
PubMed1999

Homogeneous Cell- and Bead-Based Assays for High Throughput Screening Using Fluorometric Microvolume Assay Technology.

High throughput drug screening has become a critical component of the drug discovery process. The screening of libraries containing hundreds of thousands of compounds has resulted in a requirement for assays and instrumentation that are amenable to nonradioactive formats and that can be miniaturized. Homogeneous assays that minimize upstream automation of the individual assays are also preferable. Fluorometric microvolume assay technology (FMAT) is a fluorescence-based platform for the development of nonradioactive cell- and bead-based assays for HTS. This technology is plate format-independent, and while it was designed specifically for homogeneous ligand binding and immunological assays, it is amenable to any assay utilizing a fluorescent cell or bead. The instrument fits on a standard laboratory bench and consists of a laser scanner that generates a 1 mm(2) digitized image of a 100-µmm deep section of the bottom of a microwell plate. The instrument is directly compatible with a Zymark Twistertrade mark (Zymark Corp., Hopkinton, MA) for robotic loading of the scanner and unattended operation in HTS mode. Fluorescent cells or beads at the bottom of the well are detected as localized areas of concentrated fluorescence using data processing. Unbound flurophore comprising the background signal is ignored, allowing for the development of a wide variety of homogeneous assays. The use of FMAT for peptide ligand binding assays, immunofluorescence, apoptosis and cytotoxicity, and bead-based immunocapture assays is described here, along with a general overview of the instrument and software.

Journal Article↗
PubMed1996

Effects of carboxy-terminal truncation on human lecithin:cholesterol acyltransferase activity.

Mutagenesis was carried out in human lecithin:cholesterol acyltransferase (LCAT) to generate mutants with stop codons at positions corresponding to amino acids 315, 341, 359, 375, 388, 394, and 398 of the 416-amino acid sequence of the mature enzyme protein. Deletion of the 18 terminal amino acids of the protein was without effect on LCAT phospholipase or acyltransferase activity, or the stability of the protein to denaturation at 37 degrees C. Further deletion led to loss of most of the activity, associated with a 10-fold increase in the rate of denaturation at 37 degrees C. These data indicate that the proline-rich C-terminus of LCAT is not required for effective enzyme activity. The loss of activity that accompanied deletion of residues 394-398 suggests a structural role for these residues, part of a series of predicted beta-sheet sequences in the C-terminal third of the LCAT primary sequence.

Animals↗
PubMed1993

Lecithin-cholesterol acyltransferase: effects of mutagenesis at N-linked oligosaccharide attachment sites on acyl acceptor specificity.

Site-directed mutagenesis was used to generate lecithin-cholesterol acyltransferase (LCAT) species in which individual attachment sites for N-linked oligosaccharide residues were replaced with residues that prevent the attachment of carbohydrate. Mutants at three of four sites retained significant acyltransferase activity, and phospholipase activity in the absence of cholesterol. Mutation at one site (asn272) converted LCAT to a phospholipase generating fatty acids not cholesteryl esters.

Amino Acid Sequence↗
PubMed1989

Beta-thalassemia mutations in Indonesia and their linkage to beta haplotypes.

A total of 72 chromosomes from 36 Indonesian patients, 23 with beta-thalassemia major and 13 with Hb E-beta-thalassemia, were analyzed by specific oligonucleotide hybridization after DNA amplification. Thirteen had the beta E mutation (codon 26 GAG----AAG). Of the 59-beta-thalassemic chromosomes, 32 were of the variant IVS-1 nt5 (G----C). Seven had the mutation IVS-2 nt654 (C----T), one had the mutation codon 41/42 (deletion CTTT), and one had the mutation codon 17 (AAG----TAG). Another six with the mutation IVS-1 nt1 (G----T), one with the mutation IVS-1 nt1 (G----A), four with the mutation codon 15 (TGG----TAG), one with a mutation codon 30 (AGG----ACG), and one with a mutation codon 35 (deletion C) were first identified by direct sequencing of a patient's genomic DNA followed by further hybridizing other patients' DNA with the appropriate oligonucleotide probes. Five did not carry the common mutations previously described in Asian populations. The four most prevalent mutations encountered made up 83% of the total number of beta-thalassemic chromosomes studied. The most common mutation, IVS-1 nt5 (G----C), was mostly associated with two different haplotypes.

Blotting, Southern↗
PubMedDate not supplied

Retention and use of cardiopulmonary resuscitation skills in parents of infants at risk for cardiopulmonary arrest.

Policies of most neonatal intensive care units include teaching cardiopulmonary resuscitation (CPR) to parents or other caretakers prior to infant hospital discharge. However, little is known about CPR skills retention in this population or the outcome of parents' use of CPR. This is a study to measure CPR skills 6 months following CPR training to identify characteristics predicting successful performance and to determine if parents used CPR. A sample of 100 parents or related caretakers of infants at risk for an out-of-hospital respiratory or cardiac arrest 6 months following CPR training were asked to demonstrate CPR on an infant mannequin and 94 agreed to participate. Although they were excluded from the study if they had a CPR course within the past 2 years, 37% had taken CPR sometime in the past. Only one third of participants (n = 31, 33%) were able to perform satisfactory CPR. Those who demonstrated satisfactory CPR skills were more likely to have had previous CPR training and to have experienced higher levels of social support at the time of training than those who achieved unsatisfactory CPR performance ratings (p < .05). A logistic regression analysis revealed previous CPR training, social support, and level of anxiety at time of CPR training to be the most important predictors of CPR skills retention. Seven parents reported using CPR to resuscitate their infant who had suffered a respiratory arrest. All seven were successful. CPR skills decay is significant for caregivers of infants at high risk for cardiopulmonary arrest. Parents should be encouraged to review the steps of CPR frequently and to attend refresher classes. A significant proportion of parents of infants hospitalized in the neonatal intensive care unit are called upon to use CPR and are able to use it appropriately.

Adult↗