Search PubMed⌕ Search

Biomedical subjects

R F Lee

Publications and source records attributed to R F Lee.

At least 37 records · Page 2Linked to original sources
PubMed1994

The diverged copy of the citrus tristeza virus coat protein is expressed in vivo.

Nucleotide sequence analysis of a portion of the citrus tristeza closterovirus (CTV) genome revealed an open reading frame immediately upstream of the coat protein gene that can encode a protein with a calculated M(r) of 27,360 (p27). The deduced amino acid sequence indicated that this putative nonstructural gene product is highly homologous to the coat protein. To investigate whether p27 was expressed in CTV-infected plants, a fusion protein of p27 produced in Escherichia coli was used to raise polyclonal antibodies. Western blot analysis using the p27 antibodies indicated that p27 is expressed in CTV-infected citrus, but not in uninfected plants. Tissue fractionation studies revealed that p27 accumulates in cell wall enriched fractions.

Amino Acid Sequence↗
PubMed1994

Pressure effects and thermal stability of myosin rods and rod minifilaments: fluorescence and circular dichroism studies.

In the present study hydrostatic pressure was applied upon both skeletal myosin rod molecules and rod minifilaments to learn more of the intra- and intermolecular interaction behavior of myosin. Applied pressure disassembled the rod minifilaments into individual rod molecules and dissociated each myosin rod molecule into two chains of alpha-helix. The dissociation and disassembly profiles of these systems were obtained by measuring their fluorescent anisotropy under pressure. The mid-disassembly pressure of rod minifilaments at 0.4 mg/mL concentration was 430-490 bar. However, dissociation of two helical strands of rod molecules occurred at a much higher pressure, with a mid-disassembly pressure of 1300 bar at this concentration. These results indicate that the intramolecular interactions occurring between two alpha-helical chains of a rod molecule are much more stable under pressure than the intermolecular interactions that occur among rod molecules in a minifilament. The regions in the rod molecules involved in filament assembly were investigated through usage of both the intrinsic fluorescence of tryptophan residues and the extrinsic fluorescence of 6-acryloyl-2-(dimethylamino)naphthalene (acrylodan) labeled cysteine residues. The blue spectral shifts upon minifilament formation suggest the participation of both light meromyosin (LMM) and subfragment-2 (S-2) regions of myosin rods in the filament formation. Profiles of thermal unfolding of myosin rod molecules and rod minifilaments were obtained by circular dichroism measurement. The multiple transitions exhibited upon unfolding profiles indicated the presence of more than one structural domain, each correlating with a cooperative transition. The domain transitional temperatures were found to be 1-4 degrees C higher for rods in minifilaments than those for rod molecules in a solution of similar ionic composition, indicating that all structural domains are involved in filament assembly. Furthermore, the domain transitional temperatures for rod molecules in a buffer containing 0.6 M NaCl were 6-8 degrees C higher than those for rod molecules in 5 mM sodium pyrophosphate buffer, suggesting that each structural domain of a rod molecule becomes stabilized at 0.6 M NaCl solution.

Animals↗
PubMed1994

Lipophorin as a lipid carrier in the hemolymph of the horseshoe crab, Limulus polyphemus.

A high-density lipoprotein (HDL) was obtained from the hemolymph of Limulus polyphemus in yields generally less than 30 micrograms/ml by ultracentrifugal flotation. SDS-PAGE revealed two apolipoproteins with masses similar to those of apolipophorins (apoLp-I, 265 +/- 14 kDa; apoLp-II, 89 +/- 6 kDa). Lipid composition was different from both insect lipophorin and crustacean HDL, and showed less diacylglycerols than triacylglycerols (3.8% and 36.2% of total lipids, respectively). Since Limulus polyphemus is closely related to precambrian chelicerates, our results confirm that lipophorin was present early in the evolution of arthropods.

Animals↗
PubMed1993

Molecular characterization of a structural epitope that is largely conserved among severe isolates of a plant virus.

Direct molecular evidence was obtained for the critical role of a single amino acid residue in a structural epitope distinguished by the monoclonal antibody MCA-13, which reacts selectively with severe isolates of citrus tristeza virus (CTV). Different CTV isolates cause a wide range of symptoms in the diverse citrus species they affect. Severe symptoms include decline, stem pitting, and seedling yellows. Plants infected by mild isolates are essentially symptomless. The monoclonal antibody MCA-13, which discriminates severe isolates from mild isolates of the virus, was used to map its epitope on the coat protein of CTV. A diverse group of coat protein genes of geographically and biologically distinct CTV isolates which are either MCA-13-reactive or MCA-13-nonreactive was cloned and sequenced. A series of mutant coat protein genes was constructed through oligonucleotide-directed, site-specific mutagenesis. The reactivity of the wild-type and mutant coat proteins expressed in Escherichia coli was evaluated by Western blotting using MCA-13 and polyclonal antibody prepared to CTV-coat protein. A single nucleotide alteration resulting in a Phe-->Tyr mutation at position 124 of the coat protein abolished the MCA-13 reactivity of a severe isolate, whereas a Tyr-->Phe mutation at the same site conferred MCA-13 reactivity on the coat protein of a previously nonreactive mild isolate of CTV.

Amino Acid Sequence↗
PubMed1991

Detection of Chlamydia trachomatis endocervical infection in asymptomatic and symptomatic women: comparison of deoxyribonucleic acid probe test with tissue culture.

A deoxyribonucleic acid probe assay (PACE 2, Gen-Probe, San Diego) was compared with a standard tissue culture method for detection of Chlamydia trachomatis endocervical infection in both asymptomatic and symptomatic women. The results of the probe test were expressed as a ratio of relative light units of the specimen per relative light units of the cutoff recommended by the manufacturer. Samples with sample/cutoff ratios near 1.0 were repeated until two or more consistent ratios were obtained. A total of 426 specimens were obtained, with an overall disease prevalence of 10.1%. Of the 426 specimens examined, seven (1.6%) were near the cutoff and were retested. The results of 426 samples with matching cultures indicated that the manufacturer's discrete cutoff was adequate for results determination. The deoxyribonucleic acid probe test was essentially equivalent to standard tissue culture in terms of sensitivity, specificity, and positive and negative predictive values in a low-prevalence patient population.

Binding, Competitive↗
PubMed1991

Development of a dot-immunobinding assay for detection of citrus tristeza virus.

The dot-immunobinding assay (DIBA) was adapted for detection of citrus tristeza virus (CTV) and compared with DAS-ELISA and DAS-indirect ELISA. DIBA was easy to perform and as sensitive as either ELISA procedure for CTV diagnosis. The entire test could be performed in 2-3 h using polyclonal antibodies, with minimal laboratory equipment. Three different polyclonal antibodies gave a strong positive reaction with 12 selected CTV isolates; however, each serum had to be cross-absorbed with sap from healthy plants before use. The broad spectrum 3DF1 monoclonal antibody reacted with most of the CTV isolates. The MCA-13 strain-specific monoclonal antibody was specific for most severe CTV isolates. As blocking agents, 3% bovine serum albumin (BSA), 3% gelatin, 0.5% non-fat dry milk or 5% Triton X-100 gave an adequate white background on the nitrocellulose membranes and permitted discrimination between infected and healthy samples. However, 3% gelatin gave the best contrast between green for the healthy samples, and purple color for infected samples.

Citrus↗
PubMed1991

Serological techniques for detection of citrus tristeza virus.

Citrus tristeza virus (CTV) is the most economically important virus disease of citrus. In the last ten years, remarkable progress has been achieved in the development and improvement of new serological methods for CTV detection so that serology has become a dependable tool for many research, extension and regulatory purposes worldwide. CTV-specific polyclonal antisera and monoclonal antibodies have been developed in different research laboratories and used extensively in a wide range of different studies. This review describes the diverse serological methods developed for CTV detection and analyzes the advantages, disadvantages, relative sensitivity, applications, and present status of each method.

Citrus↗
PubMed1991

Comparison of Gen-Probe DNA probe test and culture for the detection of Neisseria gonorrhoeae in endocervical specimens.

A 2-h nonisotopic DNA probe assay for the direct detection of Neisseria gonorrhoeae in urogenital specimens has recently been modified (PACE 2; Gen-Probe, San Diego, Calif.). The new assay format was developed to increase the sensitivity of the assay and simplify procedural steps. In this study, the new DNA probe test was compared with a culture reference method for the detection of N. gonorrhoeae in endocervical specimens. The results of the DNA probe test were expressed as a ratio of relative light units (RLU) of the specimen/RLU of the cutoff recommended by the manufacturer. All patient samples with sample RLU/cutoff RLU ratios less than 0.7 were interpreted as negative, and ratios greater than 2.0 were interpreted as positive for gonorrhea. Samples with sample RLU/cutoff RLU ratios between 0.7 and 2.0 were repeated until two or more consistent negative or positive ratios were obtained. A total of 469 specimens were tested with an overall disease prevalence of 6.1%. Of the 469 patients tested, 5 specimens (1.0%) fell in this borderline region and were retested. If the manufacturer's recommended cutoff value had been used, the original DNA probe results would have resulted in two false-positives. Our data were analyzed for both symptomatic (prevalence, 11.7%) and asymptomatic (prevalence, 2%) women. The study indicated that with our modification of the manufacturer's endpoint interpretation, the DNA probe test was essentially equivalent to the culture method in terms of sensitivity, specificity, and positive and negative predictive values in both symptomatic and asymptomatic patient populations. The new DNA probe test can serve as a suitable screening and diagnostic test for the diagnosis of gonorrheal genital infections in women. Additionally, it offers the advantages of rapid turnaround time and ease of use and allows simultaneous testing for Chlamydia trachomatis on the same specimen.

Bacteriological Techniques↗
PubMed1990

Metaphoric competence in children with learning disabilities.

Metaphoric competence was examined in two groups of children with learning disabilities and one group of nondisabled peers ranging in age from 9-0 to 11-0 years. There were five girls and seven boys in each group. One group of students with learning disabilities had a history of spoken language impairment and the other group did not. Subjects were administered three verbal metaphor tasks (comprehension, preference, and completion) and a visual metaphor task, the Metaphor Triads Task (MTT). The three verbal metaphor tasks were administered in three contexts: (a) sentence, (b) story, and (c) story plus visual (pictorial) support. The group with a history of language impairment consistently performed more poorly on the metaphor tasks than the group without a history of language impairment, who, in turn, performed more poorly than the nondisabled children on all but the MTT. Context variations had no effect on children's performance. Theoretical and clinical implications will be discussed.

Aphorisms and Proverbs as Topic↗
PubMed1989

Mutations in two regions upstream of the A gamma globin gene canonical promoter affect gene expression.

Two regions upstream of the human fetal (A gamma) globin gene, which interact with protein factors from K562 and HeLa nuclear extracts, have functional significance in gene expression. One binding site (site I) is at a position -290 to -267 bp upstream of the transcription initiation site, the other (site II) is at -182 to -168 bp. Site II includes the octamer sequence (ATGCAAAT) found in an immunoglobulin enhancer and the histone H2b gene promoter. A point mutation (T----C) at -175, within the octamer sequence, is characteristic of a naturally occurring HPFH (hereditary persistence of fetal hemoglobin), and decreases factor binding to an oligonucleotide containing the octamer motif. Expression assays using a A gamma globin promoter-CAT (chloramphenicol acetyl transferase) fusion gene show that the point mutation at -175 increases expression in erythroid, but not non-erythroid cells when compared to a wild-type construct. This correlates with the actual effect of the HPFH mutation in humans. This higher expression may result from a mechanism more complex than reduced binding of a negative regulator. A site I clustered-base substitution gives gamma-CAT activity well below wild-type, suggesting that this factor is a positive regulator.

Base Sequence↗
PubMed1989

Analysis of possible repressor elements in the 5'-flanking region of the human beta-globin gene.

Human beta-globin gene expression is confined predominantly to the adult with little or no expression of this gene occurring during embryonic or fetal life. The lack of expression of this gene in embryonic and fetal erythroid tissue could be due to the absence of required positive regulatory factors in these cells or the presence of negative regulatory factors which prevent expression of the adult globin gene. To test the repressor model, we have used a gel electrophoretic mobility shift assay to identify regions in the human beta-globin gene which bind proteins found in K562 cells, a cell line that expresses embryonic and fetal globins but not adult beta-globin. DNA fragments comprising the entire human beta-globin gene were assayed using nuclear proteins from K562 cells, and four regions were found that bind proteins. These are located within the 5'-flanking region, within the first and second introns, and at the 3'-flanking region of the gene. Previous studies have suggested the presence of potential repressor sites 5' of exon 2. For this reason, we examined whether the lack of the binding regions in the 5'-flanking sequence allow expression of the human beta-globin gene in transgenic mice during embryonic life. beta-globin gene expression was confined to adult life, indicating that if a transcriptional repressor is responsible for inactivating this gene in embryonic tissue, it is not regulated solely by sequences upstream from -122 bp in the 5'-flanking region of the human beta-globin gene.

Animals↗
PubMed1989

Sites I and II upstream of the A gamma globin gene bind nuclear factors and affect gene expression.

Five broad regions in and near the A gamma globin gene specifically bind proteins in K562 nuclear extracts. Two are located 5' of the gene, one between -1349 and -1094, and the other between -384 and +52. Each of the two introns has a binding region, +184 to +284 and +930 to +1209. The fifth binding region is within the A gamma globin gene enhancer, a 750 bp Hind III fragment located 3' to the gene. The nuclear factors which bind to any or all of these regions may be important for control of regulation of the A gamma globin gene. Since HPFH point mutations occur at -175, -196 and -202, we investigated K562 nuclear factor binding to this region more closely. Footprints were obtained for two binding sites, one from -168 to -185 (site II) and the other from -267 to -293 (site I). Site II contains an octamer sequence (ATGCAAAT) important for protein binding and expression of the histone H2b and immunoglobulin genes. The HPFH mutation at -175 changes the T in the octamer sequence to a C. Site I is upstream of the bases affected by HPFH mutations. A A gamma globin gene promoter - CAT reporter fusion gene was constructed with a clustered - base substitution of site I or a T----C point mutation at -175. The mutation in site I decreases CAT expression 20X compared to wild-type in erythroid and non-erythroid cells. Site I probably binds a positive regulator. The T----C change at -175 increases expression 2-3X over wild-type in erythroid cells, but not in non-erythroid cells. This increase correlates with the effect of the naturally occurring HPFH, and may result from decreased binding of a negative effector, and/or increased positive factor binding.

Base Sequence↗
PubMed1988

Effect of preexposure to dietary benzo[a]pyrene (BP) on the first-pass metabolism of BP by the intestine of toadfish (Opsanus tau): in vivo studies using portal vein-catheterized fish.

The effect of preexposure of fish to dietary benzo[a]pyrene (BP) on the intestinal metabolism of BP was examined in toadfish (Opsanus tau). The portal veins of toadfish were cannulated following administration of radiolabeled BP to the intestinal lumen. Because these fish lack a lymphatic vessel system, the portal vein is the sole route by which BP and its metabolites enter the circulation. In fish preexposed to dietary BP (10 mg BP/kg food), the radioactivity entering the portal vein was almost entirely (ca. 90%) BP metabolites. In fish fed a laboratory control diet, a smaller percentage (ca. 60%) of the radioactivity entering the portal vein was in the form of BP metabolites. The enhanced efficiency of the intestines of preexposed fish in metabolizing BP appears to be a result of induction of intestinal aryl hydrocarbon hydroxylase (AHH) activity. Intestinal microsomal AHH activities in control and preexposed fish were 0.033 +/- 0.032 and 0.320 +/- 0.060 nmol.min-1.mg-1, respectively. Gel filtration of portal vein plasma indicated differences in the roles of plasma proteins in transporting BP and BP metabolites. Native BP was associated primarily with the high density lipoproteins, whereas organic-soluble BP metabolites were associated primarily with serum albumin fractions. A large percentage of BP metabolites was recovered as water-soluble conjugates. These studies indicate that in fish, the intestine can be an important organ involved in dietary BP metabolism.

Animals↗
PubMed1988

Genetic profile of the transcriptional signals from the adenovirus major late promoter.

Identical functional profiles were obtained for in vivo and in vitro transcription assays of more than 30 site-directed point mutants within the adenovirus major late promoter. The functional limits of the functional regions encompassing upstream promoter element are defined (-51 to -61), as well as a region around the transcription start site (-1 to +1), flanked by regions insensitive to sequence alterations.

Adenoviridae↗
PubMed1987

The purification and characterization of glutathione S-transferase from the hepatopancreas of the blue crab, Callinectes sapidus.

High glutathione S-transferase activity was found in the cytosol of F-cells from the hepatopancreas of the blue crab (Callinectes sapidus). Purification of glutathione S-transferase from hepatopancreas extracts by Sephadex G-200, DEAE-Sephacel, and chromatofocusing resulted in the isolation of two isozymes with isoelectric points of 5.9 and 5.7, as determined by analytical isoelectric focusing. Using 1-chloro-2,4-dinitrobenzene as the substrate the specific activities of the two purified isozymes were 222 and 182 mumol/min/mg, respectively. There was no evidence for basic transferase isozymes. In addition to 1-chloro-2,4-dinitrobenzene the purified glutathione S-transferase isozymes showed activity with p-nitrophenyl acetate, p-nitrobenzyl chloride, bromosulfophthalein, and benzopyrene oxide. Thus, both substitution and addition reactions associated with vertebrate glutathione S-transferase were found in the crab transferases. There was no when ethacrynic acid, methyl iodide, trans-4-phenyl-3-buten-2-one, 1,2-epoxy-(p-nitrophenoxy)propane, cumene hydroperoxide, and t-butyl hydroperoxide were used as substrates. The lack of peroxidase activity is of interest since this activity is commonly found in vertebrate transferase isozymes. The two transferases had a dimeric Mr of 40,800 with similar amino acid compositions and similar kinetic parameters (Vmax, Km, and pH maxima) with 1-chloro-2,4-dinitrobenzene as substrate. The two transferases could be distinguished by their isoelectric points, molecular mass of the monomers (22,300 for GST 1 and 22,300 and 22,400 for GST 2), and different inhibitor mechanisms with hematin and bromosulfophthalein.

Amino Acids↗
PubMed1987

Dietary fat inhibits the intestinal metabolism of the carcinogen benzo[a]pyrene in fish.

Following the intestinal absorption of dietary benzo[a]pyrene (BP) by the killifish, this compound becomes incorporated along with dietary triglycerides into membrane-bound fat vacuoles within the intestinal epithelial cell (1985. J. Lipid Res. 26: 428-434). These vacuoles, arising from the smooth endoplasmic reticulum, are important transient structures involved in both the uptake and metabolism of dietary BP and, presumably, other lipophilic toxicants as well. In the present study we used subcellular fractions isolated from the intestines of spot (Leiostomus xanthurus), a teleost fish, to study factors that influence the metabolism of BP in a fat vacuole/microsomal system. Triglyceride-solubilized BP is capable of diffusion from fat vacuoles to microsomal enzymes. Increases in the concentration of fat vacuoles decrease the availability of BP to microsomal BP hydroxylase. The effect of fat vacuoles on the activity of BP hydroxylase becomes more pronounced as the concentration of BP in our test system decreases. Addition of cytosolic glutathione transferases to the fat vacuole/microsomal system enhances the activity of BP hydroxylase. Examination of binding of 3H-labeled BP to killifish (Fundulus heteroclitus) intestinal cytosolic proteins in vivo indicated that a large fraction of the radioactivity was associated within glutathione transferase. These results suggest that dietary fat inhibits metabolism of low levels of BP in the intestine. A consequence of this would be greater exposure of peripheral tissues to dietary carcinogens.

Animals↗
PubMed1985

Word, syllable, and sound awareness in language-disordered children.

The present investigation evaluated language-disordered children's metalinguistic awareness of words, syllables, and sounds. Subjects were 15 language-disordered children matched for mental age to 15 normally developing children and for language age to another 15 normally developing children. In the first task, children were asked to divide sentences, bisyllabic words, and monosyllabic words into smaller units. In the second task, children were asked several questions designed to assess their word awareness. The language-disordered children performed significantly poorer than both groups of normally developing children in dividing sentences and words. The language-disordered children also did not show the same level of responses to the word-awareness questions as the normally developing children. These findings indicate that language-disordered children's metalinguistic deficit is not limited to difficulty making grammatical judgments. Importantly, these disordered children's lack of word, syllable, and sound awareness places them significantly at risk for future academic difficulties, in particular, learning how to read.

Awareness↗