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Biomedical subjects

J Leong

Publications and source records attributed to J Leong.

At least 37 records · Page 2Linked to original sources

Potential for improvement in radiation therapy.

A successful strategy for improving the efficacy of radiation therapy has been to improve dose distribution, that is, reduce treatment volume toward target volume. This is so as the smaller treatment volume has permitted a higher dose to the target (hence a high tumor control probability) and a lesser volume of non-target tissues being irradiated (consequently a reduced frequency and severity of treatment related morbidity). There are in place several important means for further improvements in dose distributions. These include: (a) 3D graphic reconstruction of the affected part with definition of the position of the tumor vis-a-vis the adjacent normal structures; (b) explicit inclusion in the treatment plan of the uncertainty band around each isodose contour; (c) on-line contrast enhanced visual monitoring of the target tissue during the individual treatment session; (d) gating of treatment so as to reduce the impact of patient motion on the needed treatment volume; (e) use of computer control systems to execute the treatment; and (f) use of treatment methods which achieve a reduced treatment volume. In an examination for sites for which treatment volumes might be decreased by a substantial factor we have compared treatment volumes for radical surgical and radiation therapy. Results are presented for carcinomas of the cervix (Stage IB), breast (Stage II), floor of mouth (Stage II). We describe a system developed here for on-line visual monitoring of the tissues covered by the treatment field. Brief descriptions are given of results of low LET charged particle radiation therapy and of intraoperative electron beam therapy. Also, the program developed here to use computer graphic techniques to display tumor and normal structures and isodose countours with uncertainty bands around each contour is mentioned.

Combined Modality Therapy↗

Functional role of cysteine-146 in Escherichia coli thymidylate synthase.

Analysis of mutant Escherichia coli thymidylate synthases (EC 2.1.1.45) with various amino acids substituted for cysteine at position 146 revealed the cysteine to be involved in the binding of 2'-deoxyuridylate as well as initiating the catalytic process. The substitution of a serine or alanine residue at position 146 did not appreciably alter the binding affinity for 2'-deoxyuridylate but the serine mutant enzyme was less active by a factor of 5000, whereas the alanine mutant enzyme was catalytically inactive. In contrast, the substitution of a glycine or threonine at position 146 created inactive enzymes with higher 2'-deoxyuridylate dissociation constants. The dissociation constant values for 2'-deoxyuridylate were used to estimate the overall contribution of the side chain of the amino acid at position 146 to substrate binding. The results suggested that the side chains of cysteine, alanine, and serine make nonspecific but effective van der Waals contacts with 2'-deoxyuridylate, thereby contributing about 0.82 kcal.mol-1 (1 cal = 4.184 J) to the apparent binding energy of the substrate.

Catalysis↗

Magnetic resonance imaging in multiple sclerosis: decreased signal in thalamus and putamen.

High-field strength (1.5 Tesla) magnetic resonance imaging in 15 patients with multiple and extensive white-matter lesions and clinically definite multiple sclerosis delineated a previously undescribed finding of abnormally decreased signal intensity on T2-weighted images in the thalamus and putamen. The decreased signal intensity (preferential decreased T2 relaxation time) is most likely to be related to abnormally increased iron accumulation causing local magnetic field heterogeneities.

Humans↗

Optimization of beam weights under dose-volume restrictions.

A basic problem in treatment planning is the selection of weights for a set of beams which will yield the largest tumor dose under constraints limiting the doses received in specified fractions of different normal tissue structures. This report describes a method for formulating and solving this optimization problem as a combinatorial linear program. An illustration is provided by a problem in planning treatment of a thoracic tumor, in which no more than 1/2 or 2/3 of the lung is permitted to receive greater than 20 Gy and no part of the spinal cord allowed to receive greater than 45 Gy. The optimization technique was applied to this example to determine how the maximum tumor dose is affected by changes in the normal tissue constraints and the addition of a tumor dose homogeneity restriction. The linear programming technique yielded a rigorous and efficient determination of the beam weights for the thoracic plan considered. An exhaustive specification of all the underlying linear programs allows problems of moderate dimensions to be solved, while developments in mathematical programming and computer processing suggest approaches to problems of greater complexity.

Humans↗

Implementation of random positioning error in computerised radiation treatment planning systems as a result of fractionation.

Fractionation is widely employed in radiation therapy. The number of fractions N can vary from 1 to more than 25. Associated with this multifraction treatment is the inability to reposition patients exactly from fraction to fraction or from patient to patient. The effect of this positioning error is analysed within the context of computerised treatment planning (dose calculation). A simple method is described in which this analysis can be easily incorporated into most computerised two-dimensional treatment planning systems. The result is a 'modified' isodose plan in which fluctuation in patient positioning from fraction to fraction is taken into account. The effect of N on dose calculation is further analysed. A procedure to express dose predictions on paper plan due to the finite value of N is described in terms of the uncertainty in dose predictions and the most probable dose predictions.

Humans↗

Identification and purification of a recombinant Treponema pallidum basic membrane protein antigen expressed in Escherichia coli.

A recombinant plasmid designated pLVS3 previously was described that harbored a 14-kilobase insert of Treponema pallidum genomic DNA. Escherichia coli maxicells programmed with this plasmid synthesized three treponemal protein antigens of molecular weights 39,000, 35,000, and 25,000 (39K, 35K, and 25K proteins, respectively). In this study, a detailed deletion analysis of pLVS3 demonstrated that the genetic information for all three protein antigens is contained within a 1.5-kilobase EcoRI-HpaI restriction fragment. The DNA sequence of this fragment revealed a single open reading frame of 361 codons that most likely encodes a signal peptide-bearing precursor to the 39K protein that can be transiently detected in E. coli maxicells. Evidence indicated that the 35K and 25K protein antigens are derivatives of the larger protein and are only produced in maxicells. A significant elevation in expression of the 39K treponemal protein antigen in E. coli was obtained by using the E. coli lpp and lac promoters and a genetic construction in which the signal peptide and first four residues of the "mature" 39K protein were replaced by six amino acids encoded by the vector. This hybrid protein exhibited an unusually high pI, which greatly facilitated its purification to homogeneity. By using antibody prepared against the hybrid protein, the native treponemal protein counterpart, also of molecular weight 39,000, was identified as a membrane component of T. pallidum. Since the native protein also exhibited a net positive charge, it has been designated the T. pallidum basic membrane protein.

Antigens, Bacterial↗

Nucleotide sequence of a cDNA clone carrying the glycoprotein gene of infectious hematopoietic necrosis virus, a fish rhabdovirus.

The nucleotide sequence of the mRNA encoding the glycoprotein of infectious hematopoietic necrosis virus was determined from a cDNA clone containing the entire coding region. The G-protein cDNA is 1,609 nucleotides long (excluding the polyadenylic acid) and encodes a protein of 508 amino acids. The predicted amino acid sequence was compared with that of the glycoprotein of the Indiana and New Jersey serotypes of vesicular stomatitis virus and with the glycoprotein of rabies virus, using a computer program which determined optimal alignment. An amino acid identity of approximately 20% was found between infectious hematopoietic necrosis virus and the two vesicular stomatitis virus serotypes and between infectious hematopoietic necrosis virus and rabies virus. The positions and sizes of the signal sequence and transmembrane domain and the possible glycosylation sites were determined.

Amino Acid Sequence↗

Structure of pseudobactin A214, a siderophore from a bean-deleterious Pseudomonas.

Bean-deleterious Pseudomonas A214 produced the extracellular yellow-green, fluorescent siderophore [microbial iron(III) transport agent] pseudobactin A214 under iron-limiting conditions. Pseudobactin A214 has a molecular formula of C46H64N13O22 and a molecular mass of 1151 g/mol. Pseudobactin A214 contained an N-blocked linear octapeptide with the amino acid sequence Ser-Ala-Gly-Ser-Ala-threo-beta-OH-Asp-L-allo-Thr-N delta-OH-Orn with a yellow-green, fluorescent quinoline derivative attached via an amide bond to the amino terminus. A succinamide group was linked to carbon 3 of the quinoline derivative. Sequencing was accomplished by two-dimensional NMR spectroscopy and by Edman degradation of smaller peptides obtained from partial acid hydrolysis. Since pseudobactin A214 was not affected by nonspecific proteolytic enzymes, it might contain D-amino acids. The three bidentate iron-(III)-chelating groups consisted of a 1,2-dihydroxy aromatic group in the quinoline chromophore, an alpha-hydroxy acid group present as beta-hydroxyaspartic acid, and a hydroxamate group derived from N delta-acetyl-N delta-hydroxyornithine. The chemical structure of pseudobactin A214 is remarkably similar to those of pseudobactin and pseudobactin 7SR1, the siderophores of plant growth promoting and plant-deleterious Pseudomonas B10 and Pseudomonas 7SR1, respectively.

Amino Acid Sequence↗

Iron transport-mediated antagonism between plant growth-promoting and plant-deleterious Pseudomonas strains.

Both plant growth-promoting Pseudomonas B10 and its yellow-green, fluorescent iron transport agent (siderophore) pseudobactin enhance potato growth and biologically control certain soil-borne fungal diseases in part by depriving specific root-colonizing endemic microorganisms including phytopathogens of iron(III), thus inhibiting their growth. The present study examines this mode of iron deprivation. The growth inhibition of certain bean-deleterious fluorescent pseudomonads by specific bean-beneficial fluorescent pseudomonads is due in part to the inability of susceptible strains to utilize siderophores from beneficial strains to transport iron(III). Conversely, deleterious strains which were able to utilize siderophores from beneficial strains were not inhibited. The ability of a given pseudomonad to utilize another pseudomonad's siderophore may depend upon its possessing a specific outer membrane receptor protein for that pseudomonad's ferric siderophore. Siderophore-mediated competition for iron in microbial systems appears to be a widespread phenomenon.

Binding, Competitive↗

Cloning of the gene coding for the outer membrane receptor protein for ferric pseudobactin, a siderophore from a plant growth-promoting Pseudomonas strain.

Plant growth-promoting Pseudomonas B10 produces its yellow-green, fluorescent siderophore (microbial iron transport agent) pseudobactin under iron-limiting conditions. A structural gene encoding the 85,000-Da putative outer membrane receptor protein for ferric pseudobactin was identified in a gene bank from Pseudomonas B10 prepared with the broad host-range conjugative cosmid cloning vector pLAFR1. Transposon Tn5 mutagenesis of recombinant plasmid pJLM300 localized the functional gene to a region of approximately 2.4 kilobases consistent with the apparent molecular weight of the receptor protein. Mobilization of pJLM300 into Pseudomonas A124 and A225, whose growth was inhibited by Pseudomonas B10 or pseudobactin, rendered these strains no longer susceptible to iron starvation by pseudobactin because they were now able to transport ferric pseudobactin. Pseudobactin biosynthetic genes flanked this receptor gene on both sides and were on separate operons. Transposon Tn5 insertion mutants of Pseudomonas B10 lacking this receptor protein were generated by a marker exchange technique and were defective in ferric pseudobactin transport. Such mutants could be complemented in trans by pJLM300. The production of pseudobactin, the receptor protein, and four other outer membrane proteins in Pseudomonas B10 was coordinately regulated by the level of intracellular iron.

Bacterial Proteins↗

Use of digital fluoroscopy as an on-line verification device in radiation therapy.

We have applied digital fluoroscopy technology to the radiation therapy environment, and developed a real-time fluorescent dosemeter (RFD). Our aim was to utilise this system as an on-line treatment verification device. We have found that the system behaves basically as a large array (512 X 512) of dosemeters. Using this device, instantaneous information about the actual treatment beam can be obtained simultaneously with the commencement of irradiation. As an array of dosemeters, it provides a detailed description of the dose distribution (exit dose) throughout the treatment. Furthermore, by applying image enhancement techniques to this measured information, good quality portal images are produced instantaneously and continuously during the entire irradiation for visual evaluation. The availability of this system should lead to improvements in precision in present dose delivery procedures.

Fluoroscopy↗

Accuracy of radiation field alignment in clinical practice.

We present an analysis of simulator and portal films of 71 patients. Twenty-five were analyzed retrospectively, 39 prospectively, but without changing routine filming practice, and 7 had daily portal films taken. Treatment-to-treatment variations in anatomy with respect to the field were determined by comparing sequential portal films. The standard deviation of the variations was approximately normally distributed with an average value of 3 mm independent of site and field shaping technique. Discrepancies between the portal and simulator films were greater and depended on the site of treatment. The mean worst-case discrepancy averaged over all sites was 7.7 mm; the lowest value was 3.5 mm in the head and neck region; the highest value was 9.2 mm in the thorax.

Humans↗

A method for consistent precision radiation therapy.

Using a meticulous setup procedure in which repeated portal films were taken before each treatment until satisfactory portal verifications were obtained, a high degree of precision in patient positioning was achieved. A fluctuation from treatment to treatment, over 11 treatments, of less than +/- 0.10 cm (S.D.) for anatomical points inside the treatment field was obtained. This, however, only applies to specific anatomical points selected for this positioning procedure and does not apply to all points within the portal. We have generalized this procedure and have suggested a means by which any target volume can be consistently positioned which may approach this degree of precision.

Chondrosarcoma↗

Nucleotide sequence comparison between heat-labile toxin B-subunit cistrons from Escherichia coli of human and porcine origin.

The nucleotide sequence of the LT-BH cistron (eltBH) from an enterotoxigenic Escherichia coli strain infectious for humans was determined and compared with the LT-B cistron sequence from a porcine E. coli isolate. Both cistrons were shown to comprise 375 nucleotide base pairs, and discrepancies were detected at eight positions. Of the nonhomologous base pairs, six resulted in codon changes that would lead to amino acid variations. The nucleotide sequence distal to both LT-B cistrons was also determined, and only three differences were detected in 197 base pairs. An HhaI site unique to eltBH was shown to be present in all the heat-labile (LT) genes from 31 human isolates surveyed, whereas the restriction enzyme recognition site was absent in the gene from 46 porcine E. coli isolates. The results suggest that two genetically discernable LT groups are identifiable and that the groups are also distinguishable by the isolation source (human or porcine) of the infecting E. coli strains.

Amino Acid Sequence↗

Structure of pseudobactin 7SR1, a siderophore from a plant-deleterious Pseudomonas.

When grown in iron-limiting culture medium, sugar beet deleterious Pseudomonas 7SR1 produced extra-cellularly the yellow-green, fluorescent siderophore pseudobactin 7SR1. Pseudobactin 7SR1 had a molecular formula of C46H63N13O23 and a molecular mass of 1166 g/mol. Pseudobactin 7SR1 contained a cyclic octapeptide with the amino acid sequence L-Ala-Gly-Ser-Ser-threo-beta-OH-Asp-Thr-Ser-N delta-OH-Orn. Since pseudobactin 7SR1 was not affected by nonspecific enzymes, it might contain D-amino acids. A yellow-green, fluorescent quinoline derivative is postulated to be attached via an ester bond to the serine residue following the glycine. A malamide group was attached to carbon 3 of the quinoline derivative. The three bidentate iron(III)-chelating groups consisted of an alpha-hydroxy acid group derived from beta-hydroxyaspartic acid, an omicron-dihydroxy aromatic group derived from the yellow-green, fluorescent chromophore, and a hydroxamate group derived from N delta-acetyl-N delta-hydroxyornithine. The chemical structure of pseudobactin 7SR1 is remarkably similar to that of pseudobactin, the siderophore of plant growth promoting Pseudomonas B10.

Amino Acid Sequence↗

A digital image processing system for high energy X-ray portal images.

The technique of conventional digital image processing is applied to high energy X-ray portal films. The system, which does not use state-of-the-art equipment, is analysed to determine its adequacy for such applications. Although inferior to film, notably in spatial resolution, it is found to be useful in the improvement of radiotherapy portal films.

Analog-Digital Conversion↗

Cloning of genes involved in the biosynthesis of pseudobactin, a high-affinity iron transport agent of a plant growth-promoting Pseudomonas strain.

A gene bank of DNA from plant growth-promoting Pseudomonas sp. strain B10 was constructed using the broad host-range conjugative cosmid pLAFR1. The recombinant cosmids contained insert DNA averaging 21.5 kilobase pairs in length. Nonfluorescent mutants of Pseudomonas sp. strain B10 were obtained by mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine, ethyl methanesulfonate, or UV light and were defective in the biosynthesis of its yellow-green, fluorescent siderophore (microbial iron transport agent) pseudobactin. No yellow-green, fluorescent mutants defective in the production of pseudobactin were identified. Nonfluorescent mutants were individually complemented by mating the gene bank en masse and identifying fluorescent transconjugants. Eight recombinant cosmids were sufficient to complement 154 nonfluorescent mutants. The pattern of complementation suggests that a minimum of 12 genes arranged in four gene clusters is required for the biosynthesis of pseudobactin. This minimum number of genes seems reasonable considering the structural complexity of pseudobactin.

Cloning, Molecular↗