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Biomedical subjects

R F Lee

Publications and source records attributed to R F Lee.

At least 19 recordsLinked to original sources
PubMed2002

Characterization of grapefruit plants (Citrus paradisi Macf.) transformed with citrus tristeza closterovirus genes.

Grapefruit (Citrus paradisi Macf. cv Duncan) plants were transformed with several sequences from citrus tristeza closterovirus (CTV) that varied in terms of position in the CTV genome and virus strain origin in an attempt to obtain resistant plants. The sequences included the capsid protein gene from three different strains, a nontranslatable version of the capsid protein gene, the replicase (RdRp), the minor capsid protein (p27), a highly transcribed gene of unknown function (p20) and the more conserved 3' end of the genomic RNA. Transgenic plants were generated from all of the constructs, except from the p20 and p27 genes. Southern and Western blot analyses demonstrated that stably transformed grapefruit plants were obtained and that at least some transgenes were expressed. In a first effort at virus challenge, 25 transgenic lines were graft inoculated with a severe strain of CTV. Although some transgenic plants averaged lower titers of virus than controls, there was great variability in titer in both controls and transgenic plants, and all were apparently susceptible to the virus.

Blotting, Southern↗
PubMed2001

Planar strip array (PSA) for MRI.

Parallel, spatial-encoded MRI requires a large number of independent detectors that simultaneously acquire signals. The loop structure and mutual coupling in conventional phased arrays limit the number of coils and therefore the potential reduction in minimum scan time achievable by parallel MRI tchniques. A new near-field MRI detector array, the planar strip array (PSA), is presented that eliminates the coupling problems and can be extended to a very large number of detectors and high MRI frequencies. Its basic structure is an array of parallel microstrips with a high permittivity substrate and overlay. The electromagnetic (EM) wavelength can be adjusted with the permittivity, and the strip lengths tuned to a preselected fraction of the wavelength of the MRI frequency. EM wave analysis and measurements on a prototype four-element PSA reveal that the coupling between the strips vanishes when the strip length is either an integer times a quarter wavelength for a standing-wave PSA, or a half wavelength for a travelling-wave PSA, independent of the spacing between the strips. The analysis, as well as phantom and human MRI experiments performed by conventional and parallel-encoded MRI with the PSA at 1.5 T, show that the decoupled strips produce a relatively high-quality factor and signal-to-noise ratio, provided that the strips are properly terminated, tuned, and matched or coupled to the preamplifiers. Magn Reson Med 45:673-683, 2001.

Humans↗
PubMed2000

A broadband phased-array system for direct phosphorus and sodium metabolic MRI on a clinical scanner.

Despite their proven gains in signal-to-noise ratio and field-of-view for routine clinical MRI, phased-array detection systems are currently unavailable for nuclei other than protons (1H). A broadband phased-array system was designed and built to convert the 1H transmitter signal to the non-1H frequency for excitation and to convert non-1H phased-array MRI signals to the 1H frequency for presentation to the narrowband 1H receivers of a clinical whole-body 1.5 T MRI system. With this system, the scanner operates at the 1H frequency, whereas phased-array MRI occurs at the frequency of the other nucleus. Pulse sequences were developed for direct phased-array sodium (23Na) and phosphorus (31P) MRI of high-energy phosphates using chemical selective imaging, thereby avoiding the complex processing and reconstruction required for phased-array magnetic resonance spectroscopy data. Flexible 4-channel 31P and 23Na phased-arrays were built and the entire system tested in phantom and human studies. The array produced a signal-to-noise ratio improvement of 20% relative to the best-positioned single coil, but gains of 300-400% were realized in many voxels located outside the effective field-of-view of the single coil. Cardiac phosphorus and sodium MRI were obtained in 6-13 min with 16 and 0.5 mL resolution, respectively. Lower resolution human cardiac 23Na MRI were obtained in as little as 4 sec. The system provides a practical approach to realizing the advantages of phased-arrays for nuclei other than 1H, and imaging metabolites directly.

Amplifiers, Electronic↗
PubMed2000

An analytical SMASH procedure (ASP) for sensitivity-encoded MRI.

The simultaneous acquisition of spatial harmonics (SMASH) method of imaging with detector arrays can reduce the number of phase-encoding steps, and MRI scan time several-fold. The original approach utilized numerical gradient-descent fitting with the coil sensitivity profiles to create a set of composite spatial harmonics to replace the phase-encoding steps. Here, an analytical approach for generating the harmonics is presented. A transform is derived to project the harmonics onto a set of sensitivity profiles. A sequence of Fourier, Hilbert, and inverse Fourier transform is then applied to analytically eliminate spatially dependent phase errors from the different coils while fully preserving the spatial-encoding. By combining the transform and phase correction, the original numerical image reconstruction method can be replaced by an analytical SMASH procedure (ASP). The approach also allows simulation of SMASH imaging, revealing a criterion for the ratio of the detector sensitivity profile width to the detector spacing that produces optimal harmonic generation. When detector geometry is suboptimal, a group of quasi-harmonics arises, which can be corrected and restored to pure harmonics. The simulation also reveals high-order harmonic modulation effects, and a demodulation procedure is presented that enables application of ASP to a large numbers of detectors. The method is demonstrated on a phantom and humans using a standard 4-channel phased-array MRI system.

Fourier Analysis↗
PubMed2000

Progress on strain differentiation of Citrus tristeza virus and its application to the epidemiology of citrus tristeza disease.

Citrus tristeza virus (CTV) occurs in most citrus producing regions of the world, and it is the most serious viral pathogen of citrus. With the recent establishment of the brown citrus aphid, Toxoptera citricida, its most efficient vector, on Madeira Island (Portugal) and in Florida (USA) and the countries of the Caribbean Basin, the impact of CTV is likely to increase in these regions. Since there are many strains of CTV and CTV infections frequently occur as mixtures of several strains, it is necessary to be able to distinguish the strains for regulatory purposes, disease management and epidemiology. We describe the evolution of techniques developed to detect CTV and to differentiate the individual strains, and present the results of tests using these latest methods on CTV isolates from mainland Portugal, Madeira Island and Florida. Mild and decline-inducing strains of CTV were detected in mainland Portugal and mild, decline-inducing and severe stem pitting strains on Madeira Island. In Florida we demonstrated the presence of infections that reacted with probes made against stem pitting strains not previously detected there. It is concluded that CTV presents a significant threat to citrus production in mainland Portugal, on Madeira Island and in the neighbouring countries of the Mediterranean Basin, as well as in Florida, elsewhere in the USA and throughout the Caribbean Basin, especially following the widespread establishment of T. citricida throughout the region.

Amino Acid Sequence↗
PubMed1999

Brain SPECT imaging and neuropsychological testing in coronary artery bypass patients: single photon emission computed tomography.

BACKGROUND: Cognitive deficits appear frequently after cardiac operation. While the etiology remains unclear, alterations in cerebral perfusion during cardiopulmonary bypass may be causative. Single photon emission computed tomography (SPECT) scanning utilizes a radiopharmaceutical to provide images of cerebral perfusion. We proposed to study the cerebral circulation of patients during coronary artery bypass operation employing cardiopulmonary bypass. METHODS: Thirty-five neurologically normal patients underwent preoperative SPECT brain scanning and neuropsychological testing. A second SPECT brain perfusion scan was obtained by administering the radioisotope during cardiopulmonary bypass, with subsequent scanning upon completion of the procedure. Postoperative neuropsychological testing was performed prior to discharge. RESULTS: Fourteen (40%) of patients demonstrated significant neuropsychological decline. Patients who suffered cognitive impairment were no different in demographic, general health, or surgical variables. Patients who demonstrated neuropsychological decline had significantly poorer cerebral perfusion both at baseline and during operation. CONCLUSIONS: Impaired cerebral perfusion at baseline may identify patients at risk for cognitive injury after cardiac operation. Alterations in cerebral perfusion during cardiopulmonary bypass is common, and may be a factor in neuropsychological deficits seen after cardiac operation.

Aged↗
PubMed1999

Contact leukomelanosis induced by the leaves of Piper betle L. (Piperaceae): a clinical and histopathologic survey.

BACKGROUND: In April 1997, an unusual pigmentary disorder was noticed by dermatologists in Taiwan. All patients had a history of using facial dressings with steamed leaves of Piper betle L. (Piperaceae). OBJECTIVE: Our purpose was to clarify the evolution and the origin of this unique leukomelanosis. METHODS: Fifteen patients with an unusual pigmentary disorder, who visited our clinic in September and October 1997, were asked to complete a questionnaire designed to elicit the history related to the disorder. Eight of these 15 patients underwent skin biopsies: 6 on the mottled hyperpigmented area (group A) and 2 on the hypopigmented area (group B). All 8 specimens were prepared with hematoxylin-eosin, Masson-Fontana, and S-100 stains. RESULTS: The results of the questionnaire revealed that these patients had all experienced a temporary erythematous reaction in the first few days of the use of the facial dressing, and 9 of them also complained of an accompanying stinging sensation. A bleaching effect became noticeable approximately 1 week to 1 month later. Eight patients reported that the hyperpigmentation and confetti-like hypopigmentation occurred after overexposure to the sun. In both groups, histopathologic examination revealed some melanophages in the dermis. Masson-Fontana staining of specimens from group A showed local interspersed depigmentation and hyperpigmentation in the basal epidermis and pigmentary incontinence in the dermis. This picture was different from the homogeneous depigmentation within basal epidermis in specimens from group B. In both groups, S-100 staining was negative for melanocytes in the depigmented area. CONCLUSION: The clinical course and histopathologic findings suggest that the evolution of this pigmentary disorder can be divided into 3 stages. The first stage is the immediate bleaching stage, when an irritant reaction is usually conspicuous. The second stage consists of prominent hyperpigmentation visible both grossly and microscopically. The final stage is characterized by confetti-like depigmentation. It may be induced by chemicals in the betel leaves such as phenol, catechol, and benzene derivatives, perhaps through inhibition of melanin synthesis or melanocytotoxicity.

Adult↗
PubMed1998

Distribution of total mercury and methyl mercury in water, sediment, and fish from south Florida estuaries.

Concentrations of total mercury and methyl mercury were determined in sediment and fish collected from estuarine waters of Florida to understand their distribution and partitioning. Total mercury concentrations in sediments ranged from 1 to 219 ng/g dry wt. Methyl mercury accounted for, on average, 0.77% of total mercury in sediment. Methyl mercury concentrations were not correlated with total mercury or organic carbon content in sediments. The concentrations of total mercury in fish muscle were between 0.03 and 2.22 (mean: 0.31) micrograms/g, wet wt, with methyl mercury contributing 83% of total mercury. Methyl mercury concentrations in fish muscle were directly proportional to total mercury concentrations. The relationship of total and methyl mercury concentrations in fish to those of sediments from corresponding locations was fish-species dependent, in addition to several abiotic factors. Among fish species analyzed, hardhead catfish, gafftopsail catfish, and sand seatrout contained the highest concentrations of mercury. Filtered water samples from canals and creeks that discharge into the Florida Bay showed mercury concentrations of 3-7.4 ng/L, with methyl mercury accounting for < 0.03-52% of the total mercury. Consumption of fish containing 0.31 microgram mercury/g wet wt, the mean concentration found in this study, at rates greater than 70 g/day, was estimated to be hazardous to human health.

Animals↗
PubMed1998

Annelid cytochrome P-450.

Both classes of Annelida--Polychaeta and Clitellata--have been shown to contain cytochrome P-450. The metabolism of a number of aromatic hydrocarbons, drugs and pesticides by annelids required oxygen and NADPH, and was inhibited by a variety of cytochrome P-450 inhibitors. A number of types I and II substrates bound to the cytochrome P-450 in polychaete microsomes to give typical types I and II binding spectra. These results suggest that xenobiotics in annelids are metabolized by a typical cytochrome P-450 mixed function oxygenase. In addition to xenobiotics, annelid cytochrome P-450 systems are likely to function in the biosynthesis and metabolism of sterols and hormones found in annelids, such as cholesterol, ecdysteroids and eicosanoids. The primary source of cytochrome P-450 isolated to date from annelids has been intestinal microsomes. Cytochrome P-450 concentrations in these microsomes varied from 8 to 580 pmol mg-1 of protein. The only cytochrome P-450s purified from annelids were the three isomers isolated from microsomes of the oligochaete, Lumbricus terrestris, whose molecular masses were 48,000, 51,000 and 53,000 Da. Work on the induction of cytochrome P-450 in polychaetes by exposure to polycyclic aromatic hydrocarbons or polychlorinated biphenyls has given conflicting results, since some groups found induction after such exposure, but others found no induction. One possible explanation may be exposure to natural soil and sediments inducers, e.g. plant alkaloids, during feeding. Since gene and protein sequences have yet to be carried out on the cytochrome P-450 of any annelid, the relationship of annelid cytochrome P-450s to the 74 families of P-450 so far found, remains to be carried out.

Animals↗
PubMed1997

Modulation of the antigenic reactivity of the citrus tristeza virus coat protein.

Trapping properties of a panel of monoclonal antibodies (Mabs) raised against citrus tristeza virus (CTV) were analyzed in an indirect double-antibody sandwich ELISA (I-DAS-ELISA). These antibodies had been previously assigned by serological specificity into five groups (I to V). Mabs from group V, which are directed to conformational epitopes, trapped significant amounts of virus antigen from CTV-infected plant tissue at IgG concentration above 10 ng/ml. Mabs from groups I to IV, which are directed to linear, continuous epitopes, performed poorly as coating antibodies, even at a 1 microgram/ml concentration of the IgG's, indicating that the respective linear epitopes were inaccessible. However, when Mabs from groups I to IV were combined with a small amount of Mabs from group V, a substantial increase in trapping of the CTV antigen was recorded. In this 'two antibody-binding assay' previously cryptic, linear epitopes of the CTV CP apparently became accessible to the Mabs from groups I to IV. Modulation of the antigenic reactivity of the CTV CP was also recorded upon binding of the Mabs directed to the conformational epitopes in solution. Induced exposure of the linear epitopes of the CTV CP was revealed in 'two antibody-binding assays' with pairwise combinations of different mouse Mabs and several rabbit and chicken polyclonal antisera with different serological specificities, including antisera to bacterially expressed CP fragments. This mixed coating in I-DAS-ELISA resulted in substantially increased efficiency of the virus antigen trapping by antisera produced against bacterially expressed protein fragments and an increased sensitivity of the CTV detection after optimization of the ratio between conformational and linear antibodies.

Antibodies, Viral↗
PubMed1997

Characterization of the 3' proximal gene of the citrus tristeza closterovirus genome.

The 3' proximal open reading frame (ORF 11) in the citrus tristeza virus (CTV) genome potentially encodes a protein of 209 amino acids with an estimated molecular weight of 23 kDa (p23). The p23 ORF from the severe Florida strain T36 of CTV was expressed in Escherichia coli, and the expressed protein was used to raise polyclonal antibodies in a rabbit. Using these antisera on a Western blot, a protein of expected size (23 kDa) was detected in tissue extracts from CTV-infected citrus but not from uninfected citrus. Most of the p23 protein was found in the soluble, cytoplasmic fraction. Comparison of the sequence of p23 genes from several biologically and geographically diverse CTV isolates indicated a high degree of conservation for this gene and for the RNA binding motif in particular. A cluster dendrogram of the deduced amino acid sequences correlated with the biological properties of the isolates, forming distinct groups of mild, quick decline on stem pitting-inducing isolates. Therefore it is possible that, in addition to the capsid protein gene, the p23 gene also may serve as an indicator for disease severity.

Amino Acid Sequence↗
PubMed1996

Organization of the 3'-terminal half of beet yellow stunt virus genome and implications for the evolution of closteroviruses.

The 3'-terminal half of the beet yellow stunt virus (BYSV) genome 10,545 nt, has been cloned and sequenced. The sequenced portion of the BYSV genome encompasses 10 open reading frames (ORFs) and 241 nt of the 3' untranslated region. The sequence spans, in the 5' to 3' direction, the C-terminal region of the replication-associated polyprotein gene (ORF 1a) which includes the set of motifs typical of helicases (HEL), the entire 53-kDa polymerase (RdRp) gene (ORF 1b), and genes encoding 30-kDa (ORF 2), 6-kDa (ORF 3), 66-kDa (ORF 4), 61-kDa (ORF 5), 25-kDa (ORF 6), 23.7-kDa (coat protein, CP) (ORF 7), 18-kDa (ORF 8), and 22-kDa (ORF 9) proteins. The double-stranded RNA "replicative form" of the BYSV was demonstrated to have a nontemplate G residue at the 3' terminus of the (+) strand. The RdRp of BYSV is presumably expressed via a +1 ribosomal frameshift. The five-gene module conserved among closteroviruses was identified in BYSV; it includes a gene array coding for a 6-kDa small hydrophobic protein, a 66-kDa homolog of the cellular HSP70 heat shock proteins, a 61-kDa protein, and a 25-kDa diverged copy of the CP followed by the CP gene itself. Phylogenetic analysis of the replication-associated HEL and RdRp domains as well as proteins from the five-gene module demonstrated the closest relationship between BYSV and two other closteroviruses, beet yellows (BYV) and citrus tristeza (CTV) viruses. Like CTV, the BYSV genome contains a 30-kDa protein gene between the RdRp and the 6-kDa protein genes, and like BYV it has only two genes downstream of the CP gene. The organization of the BYSV genome appears to be intermediate between BYV and CTV, which suggests that these three viruses might represent three distinct but probably close stages in the closterovirus evolution.

Amino Acid Sequence↗
PubMed1995

Multiple species of defective RNAs in plants infected with citrus tristeza virus.

Alemow (Citrus macrophylla) and sweet orange (C. sinensis) plants infected, respectively, with several Israeli and Florida isolates of the citrus tristeza virus (CTV) were found to contain multiple species of RNA molecules with features similar to defective-interfering RNAs. Northern blot hybridizations of dsRNAs extracted from serial passages of the Israeli VT isolate (CTV-VT) and from different plants infected with a single source of inoculum showed considerable variation both in the presence and in the relative abundance of the defective RNA (D-RNA) bands. The D-RNA molecules were found to be encapsidated in the CTV particles. Sequence analysis of two VT D-RNA molecules of 2.7 and 4.5 kb revealed that they were composed of two regions corresponding to 1818 and 4036 nucleotides from the 5' and 938 and 442 nucleotides from the 3' termini of the CTV-VT genomic RNA, respectively. A short (ca. 0.8 kb) nonencapsidated single-stranded positive-sense RNA species was also found in infected plants. This ssRNA, which copurified with dsRNAs, was shown by hybridization to encompass the 5'-terminal part of the CTV genome and might have an extensive secondary structure.

Base Sequence↗
PubMed1995

Complete sequence of the citrus tristeza virus RNA genome.

The sequence of the entire genome of citrus tristeza virus (CTV), Florida isolate T36, was completed. The 19,296-nt CTV genome encodes 12 open reading frames (ORFs) potentially coding for at least 17 protein products. The 5'-proximal ORF 1a starts at nucleotide 108 and encodes a large polyprotein with calculated MW of 349 kDa containing domains characteristic of (from 5' to 3') two papain-like proteases (P-PRO), a methyltransferase (MT), and a helicase (HEL). Alignment of the putative P-PRO sequences of CTV with the related proteases of beet yellows closterovirus (BYV) and potyviruses allowed the prediction of catalytic cysteine and histidine residues as well as two cleavage sites, namely Val-Gly/Gly for the 5' proximal P-PRO domain and Met-Gly/Gly for the 5' distal P-PRO domain. The autoproteolytic cleavage of the polyprotein at these sites would release two N-terminal leader proteins of 54 and 55 kDa, respectively, and a 240-kDa C-terminal fragment containing MT and HEL domains. The apparent duplication of the leader domain distinguishes CTV from BYV and accounts for most of the size increase in the ORF 1a product of CTV. The downstream ORF 1b encodes a 57-kDa putative RNA-dependent RNA polymerase (RdRp), which is probably expressed via a +1 ribosomal frameshift. Sequence analysis of the frameshift region suggests that this +1 frameshift probably occurs at a rare arginine codon CGG and that elements of the RNA secondary structure are unlikely to be involved in this process. The complete polyprotein resulting from this frameshift event has a calculated MW of 401 kDa and after cleavage of the two N-terminal leaders would yield a 292-kDa protein containing the MT, HEL, and RdRp domains. Phylogenetic analysis of the three replication-associated domains, MT, HEL, and RdRp, indicates that CTV and BYV form a separate closterovirus lineage within the alpha-like supergroup of positive-strand RNA viruses. Two gene blocks or modules can be easily identified in the CTV genome. The first includes the replicative MT, HEL, and RdRp genes and is conserved throughout the entire alpha-like superfamily. The second block consists of five ORFs, 3 to 7, conserved among closteroviruses, including genes for the CTV homolog of HSP70 proteins and a duplicate of the coat protein gene. The 3'-terminal ORFs 8 to 11 encode a putative RNA-binding protein (ORF 11), and three proteins with unknown functions; this gene array is poorly conserved among closteroviruses.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1995

Defective RNA molecules associated with citrus tristeza virus.

Preparations of single-stranded (ss) RNA extracted from particles of the Israeli VT strain of citrus tristeza virus (CTV-VT), and ss- and double-stranded (ds) RNA preparations extracted from infected Alemow (Citrus macrophylla) plants, contained a population of molecules with features that suggest that they are defective RNAs. The prototype of 2424 nt was cloned and sequenced and was found to be composed of two genomic regions corresponding to the 5' (1151 nt) and the 3' (1259 nt) termini of the genomic CTV-RNA, with two perfect direct repeats of eight nucleotides of unknown origin at the junction site. Northern hybridization analysis demonstrated that this 2.4-kb defective RNA is an abundant species among the other CTV-specific ss- and ds-RNAs in infected plants. The 2.4-kb RNA was found encapsidated by the CTV coat protein indicating that the CTV origin of assembly is located close to the 5' or 3' terminus. This is the first defective RNA to be reported for a member of the closterovirus group.

Base Sequence↗
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↗