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Biomedical subjects

D A Low

Publications and source records attributed to D A Low.

At least 19 recordsLinked to original sources
PubMed1995

Clinical implementation of a commercial multileaf collimator: dosimetry, networking, simulation, and quality assurance.

PURPOSE: Clinical implementation of multileaf collimation (MLC) includes commissioning (including leaf calibration), dosimetric measurements (penumbra, transmission, calculation parameters), shaping methods, networking for file transfer, verification simulation, and development of a quality assurance (QA) program. Differences of MLC and alloy shaping in terms of penumbra and stair-step effects must be analyzed. METHODS AND MATERIALS: Leaf positions are calibrated to light field. The resultant decrement line, penumbras, leaf transmission data, and isodoses in various planes were measured with film. Penumbra was measured for straight edges and corners, in various media. Ion chambers were used to measure effects of MLC on output, scatter, and depth dose. We maintain midleaf intersection criteria. MLC fields are set 7 mm beyond planning target volumes. After shaping by vendor software or by our three-dimensional planning system, files are transferred to the MLC workstation by means of sharing software, interface cards, and cabling. A MLC emulator was constructed for simulation. Our QA program includes file checks, monthly checks (leaf position accuracy and interlock tests), and annual review. RESULTS: We found the MLC leaf position (light field) corresponds to decrement lines ranging from 50 to 59%. Transmission through MLC (1.5-2.5%) is less than alloy (3.5%). Multileaf penumbra is slightly wider than for alloy. Relative penumbra did not increase in the lung, and composite field dosimetry exhibited negligible differences compared with alloy. Verification simulations provide diagnostic image quality hard copies of the MLC fields. Monitor unit parameters used for alloy held for MLC. DISCUSSION: Clinical implementation for MLC as a block replacement was conducted on a site-by-site basis. Time studies indicate significant (25%) in-room time reductions. Through imaging and dosimetric analysis, the accuracy of field delivery has increased with MLC. The most significant impact of MLC is the ability to increase the number of daily treatment fields, thereby reducing normal tissue dosing, which is vital for dose escalation.

Calibration↗
PubMed1995

Computer-aided design and fabrication of an electron bolus for treatment of the paraspinal muscles.

PURPOSE: Demonstrate the technology for the design, fabrication, and verification of an electron bolus used in the preoperative irradiation of a mesenchymal chondrosarcoma in the paraspinal muscle region (T8-T12), in which the target volume overlay a portion of the spinal cord, both lungs, and the right kidney. METHODS AND MATERIALS: An electron-bolus design algorithm implemented on a three dimensional (3D) radiotherapy treatment planning system designed the bolus to yield a dose distribution that met physician-specified clinical criteria. Electron doses were calculated using a 3D electron pencil-beam dose algorithm. A computer-driven milling machine fabricated the bolus from modeling wax, machining both the patient surface and the beam surface of the bolus. Verification of the bolus fabrication was achieved by repeating the patient's computed tomography (CT) scan with the fabricated bolus in place (directly on the posterior surface of the prone patient) and then recalculating the patient's dose distribution using the 3D radiotherapy treatment planning system. RESULTS: A treatment plan using a 17-MeV posterior electron field with a bolus delivered a superior dose distribution to the patient than did the same plan without a bolus. The bolus plan delivered a slightly increased dose to the target volume as a result of a slightly broader range of doses. There were significant reductions in dose to critical structures (cord, lungs, and kidney) in the bolus plan, as evidenced by dose-volume histograms (DVHs). The patient dose distribution, calculated using CT scan data with the fabricated bolus, showed no significant differences from the planned dose distribution. CONCLUSIONS: A bolus can provide considerable sparing of normal tissues when using a posterior electron beam to irradiate the paraspinal muscles. Bolus design and fabrication using the tools described in this paper are adequate for patient treatment. CT imaging of the patient with the bolus in place followed by calculation of the patient's dose distribution demonstrated a useful method for verification of the bolus design and fabrication process.

Adolescent↗
PubMed1995

Leucine-responsive regulatory protein plays dual roles as both an activator and a repressor of the Escherichia coli pap fimbrial operon.

The expression of the pap pilus operon of Escherichia coli is under a phase-variation control mechanism in which cells undergo a reversible transition between transcriptionally active (phase ON) and inactive (phase OFF) states. In this study, we explore the roles of leucine-responsive regulatory protein (Lrp) and the histone-like protein H-NS in the regulation of pap phase variation. Our data indicate that the phase OFF state results from repression of the intrinsically active papBA promoter by Lrp and H-NS, each of which can act independently as transcriptional repressors. Lrp requires pap DNA sequences upstream of the papBA promoter for its repressor activity whereas H-NS does not. In contrast, in the ON state, Lrp, in conjunction with PapI, activates pap transcription. This activation is not merely a result of alleviating the H-NS mediated repression, but induces a level of transcription that is eightfold higher than the basal level of transcription from the papBA promoter measured in the absence of both H-NS and Lrp. Analysis of Lrp activation mutants indicates that binding of Lrp to pap DNA sequences is not sufficient for transcription activation, consistent with a model in which an additional domain of Lrp interacts with the transcriptional apparatus. Together, our results show that Lrp functions as a transcriptional activator in phase-ON cells and as a repressor of basal transcription in phase-OFF cells. Because pap phase variation occurs in the absence of H-NS, it is not clear what role this regulatory protein plays in pap gene regulation.

Bacterial Outer Membrane Proteins↗
PubMed1995

A convolution-adapted ratio-TAR algorithm for 3D photon beam treatment planning.

A convolution-adapted ratio of tissue-air ratios (CARTAR) method of dose calculation has been developed at the Mallinckrodt Institute of Radiology. This photon pencil-beam algorithm has been developed and implemented specifically for three-dimensional treatment planning. In a standard ratio of tissue-air ratios (RTAR) algorithm, doses to points in irregular field geometries are not adequately modeled. This is inconsistent with the advent of conformal therapy, the goal of which is to conform the dose distribution to the target volume while sparing neighboring sensitive normal critical structures. This motivated us to develop an algorithm that can model the beam penumbra near irregular field edges, while retaining much of the speed for the original RTAR algorithm. The dose calculation algorithm uses two-dimensional (2D) convolutions, computed by 2D fast Fourier transform, of pencil-beam kernels with a beam transmission array to calculate 2D off-axis profiles at a series of depths. These profiles are used to replace the product of the transmission function and measured square-field boundary factors used in the standard RTAR calculation. The 2D pencil-beam kernels were derived from measured data for each modality using commonly available dosimetry equipment. The CARTAR algorithm is capable of modeling the penumbra near block edges as well as the loss of primary and scattered beam in partially blocked regions. This paper describes the dose calculation algorithm, implementation, and verification.

Algorithms↗
PubMed1992

Leucine-responsive regulatory protein controls the expression of both the pap and fan pili operons in Escherichia coli.

The methylation blocking factor gene (mbf) in Escherichia coli is required for specific methylation inhibition of two DNA GATC sites upstream of the papBA pilin promoter and transcriptional activation of pap. Complementation and mutational analysis using pap-lac and ilvIH-lac operon fusions indicates that the mbf gene is identical to a recently described global regulatory gene lrp (leucine-responsive regulatory protein) that acts as a positive regulator of some genes and a negative regulator of others in E. coli. DNA sequence analysis of an mbf::mTn10 insertion showed that the mbfDNA sequence was identical to lrp. Thus Lrp inhibits DNA methylation at specific GATC sites. We also show that Lrp positively regulates transcription of the fan operon, which encodes K99 pili of diarrheagenic E. coli. Purified Lrp was found to bind to DNA fragments encompassing the pap and fan promoters, which is consistent with previous results indicating that Lrp controls gene expression by binding to regulatory DNA sites. Exogenous leucine significantly reduced fan transcription and K99 pili expression, similar to results obtained with the ilvIH operon. However, pap gene expression was unresponsive to leucine, which distinguishes pap from other lrp-regulated genes whose expression is modulated by leucine.

Amino Acid Sequence↗
PubMed1992

Evidence for global regulatory control of pilus expression in Escherichia coli by Lrp and DNA methylation: model building based on analysis of pap.

Pyelonephritis-associated pilus (Pap) expression is regulated by a phase variation control mechanism involving PapB, Papl, catabolite activator protein (CAP), leucine-responsive regulatory protein (Lrp) and deoxyadenosine methylase (Dam). Lrp and Papl bind to a specific non-methylated pap regulatory DNA region containing the sequence 'GATC' and facilitate the formation of an active transcriptional complex. Evidence indicates that binding of Lrp and Papl to this region inhibits methylation of the GATC site by Dam. However, if this GATC site is first methylated by Dam, binding of Lrp and Papl is inhibited. These events lead to the formation of two different pap methylation states characteristic of active (ON) and inactive (OFF) pap transcription states. The fae (K88), daa (F1845) and sfa (S) pilus operons share conserved 'GATC-box' domains with pap and may be subject to a similar regulatory control mechanism involving Lrp and DNA methylation.

Bacterial Proteins↗
PubMed1992

Thermoregulation of the pap operon: evidence for the involvement of RimJ, the N-terminal acetylase of ribosomal protein S5.

Our previous work showed that pap pilin gene transcription is subject to a thermoregulatory control mechanism under which pap pilin is not transcribed at a low temperature (23 degrees C) (L. B. Blyn, B. A. Braaten, C. A. White-Ziegler, D. H. Rolfson, and D. A. Low, EMBO J. 8:613-620, 1989). In order to isolate genes involved in this temperature regulation of gene expression, chromosomal mini-Tn10 (mTn10) mutations that allowed transcription of the pap pilin gene at 23 degrees C were identified, and the locus was designated tcp, for "thermoregulatory control of pap" (C. A. White-Ziegler, L. B. Blyn, B. A. Braaten, and D. A. Low, J. Bacteriol. 172:1775-1782, 1990). In the present study, quantitative analysis showed that the tcp mutations restore pap pilin transcription at 23 degrees C to levels similar to those measured at 37 degrees C. By in vivo recombination, the tcp mutations were mapped to phage E4H10S of the Kohara library of the Escherichia coli chromosome (Y. Kohara, K. Akiyama, and K. Isono, Cell 50:495-508, 1987). The tcp locus was cloned by complementation, in which a 1.3-kb DNA fragment, derived from the Kohara phage, was shown to restore thermoregulation to the mTn10 mutants. DNA sequencing revealed two open reading frames (ORFs) encoding proteins with calculated molecular masses of 22.7 and 20.3 kDa. The sequence of the 22.7-kDa ORF was identical to that of rimJ, the N-terminal acetylase of the ribosomal protein S5. The gene encoding the 20.3-kDa ORF, designated g20.3 here, did not display significant homology to any known DNA or protein sequence. On the basis of Northern (RNA) blot data, rimJ and g20.3 are located within the same operon. Two of the mTn10 transposons in the thermoregulatory mutants were inserted within the coding region of rimJ, indicating that the RimJ protein plays an important role in the temperature regulation of pap pilin gene transcription. However, rimJ itself is not thermoregulated, since rimJ transcripts were detected at both 23 and 37 degrees C. Disruption of the g20.3 gene by insertion and deletion mutagenesis did not affect thermoregulation of the pap operon, suggesting that, although g20.3 lies within the same operon as rimJ, it does not play a role in thermoregulation.

Acetylation↗
PubMed1991

Effect of dimensionality of heterogeneity corrections on the implementation of a three-dimensional electron pencil-beam algorithm.

Electron beam dose distributions were calculated on a three-dimensional grid using three pencil-beam algorithms, each taking into account irregularities in field shape. The algorithms differ in that patient anatomy in either one, two, or three dimensions is used in the calculation of dose to a point. Algorithms were optimized for speed by such techniques as precalculation and storage of several quantities, reordering of pencil-beam and grid-point loops, selection of cut-off values for some calculated quantities, and invoking error function symmetries. Execution times for optimized versions of each of the algorithms as implemented on a three-dimensional treatment planning system were comparable for both the one- and two-dimensional heterogeneity correction requires an additional calculational loop over fan lines. Execution times for the three-dimensional heterogeneity correction were approximately a factor of four longer than those for the two-dimensional correction. For certain geometries, three-dimensional heterogeneity corrections were necessary to calculate dose distributions accurately, in spite of the additional cost in calculation times.

Algorithms↗
PubMed1991

Legionella pneumophila inhibits protein synthesis in Chinese hamster ovary cells.

Legionella pneumophila is a gram-negative facultative intracellular parasite that causes Legionnaires disease. To explore the interactions between L. pneumophila and host cells, we have developed a continuous cell line model of infection. We show that about 80% of Chinese hamster ovary (CHO) cells were associated with L. pneumophila after incubation for 3 h at a multiplicity of infection of 20 bacteria per cell. Within 3 to 4 h of incubation with L. pneumophila, protein synthesis of CHO cells was markedly inhibited, as shown by the reduction of incorporation of radiolabeled amino acids into proteins. L. pneumophila did not inhibit transport of amino acids or cause degradation of newly synthesized proteins in CHO cells. Cytochalasin D blocked internalization of L. pneumophila by CHO cells, yet CHO cell protein synthesis was inhibited. These results indicated that L. pneumophila could inhibit host protein synthesis from the cell exterior. L. pneumophila that had been killed with antibiotics prior to incubation with CHO cells still inhibited protein synthesis, indicating that the inhibition of CHO cell protein synthesis occurred in the absence of de novo protein synthesis by L. pneumophila.

Animals↗
PubMed1991

DNA sequences of three papA genes from uropathogenic Escherichia coli strains: evidence of structural and serological conservation.

Pyelonephritis-associated pili (Pap) are important in the pathogenesis of ascending, unobstructive Escherichia coli-caused renal infections because these surface bacterial organelles mediate digalactoside-specific binding to host uroepithelial cells. Pap are composed of many different polypeptides, of which only the tip proteins mediate specific binding. The PapA moiety polymerizes to form the bulk of the pilus structure and has been employed in vaccines despite its lack of Gal alpha(1-4)Gal receptor specificity. Animal recipients of PapA pilus-based vaccines are protected against experimental pyelonephritis caused by homologous and heterologous Gal-Gal-binding uropathogenic E. coli strains. Specific PapA immunoglobulin G antibodies in urine are correlated with protection in these infection models. The nucleotide sequences of the gene encoding PapA were determined for three E. coli clones expressing F7(1), F7(2), and F9 pili and were compared with corresponding sequences for other F serotypes. Specific rabbit antisera were employed in enzyme-linked immunosorbent assays to study the cross-reactivity between Gal-Gal pili purified from recombinant strains expressing F7(1), F7(2), F9, or F13 pili and among 60 Gal-Gal-binding wild-type strains. We present data which corroborate the concept that papA genes are highly homologous and encode proteins which exhibit greater than 70% homology among pili of different serotypes. The differences primarily occur in the cysteine-cysteine loop and variable regions and constitute the basis for serological diversity of these pili. Although there are differences in primary structures among these pili, antisera raised against pili of one serotype cross-reacted frequently with many other Gal-Gal pili of different serotypes. Furthermore, antisera raised against pili of the F13 serotype cross-reacted strongly or moderately with 52 (86%) of 60 wild-type Gal-Gal-binding E. coli strains. These data suggest that there are common immunogenic domains among these proteins. These additional data further support the hypothesis that broadly cross-protective PapA pilus vaccines for the immunoprophylaxis of pyelonephritis might be developed.

Amino Acid Sequence↗
PubMed1991

Evidence for a methylation-blocking factor (mbf) locus involved in pap pilus expression and phase variation in Escherichia coli.

Transcription of the pyelonephritis-associated pilus (pap) operon of Escherichia coli is subject to regulation by a phase variation control mechanism in which the pap pilin gene alternates between transcriptionally active (phase-on) and inactive (phase-off) states. Pap phase variation appears to involve differential inhibition of deoxyadenosine methylase (Dam) methylation of two pap GATC sites, GATC1028 and GATC1130, located in the regulatory region upstream of the papBA promoter. DNA from phase-on cells contains an unmethylated adenosine in the GATC1028 site, whereas DNA from phase-off cells contains an unmethylated adenosine in the GATC1130 site. papI and papB are two regulatory genes in the pap operon. Analysis of pap deletion mutants suggests that papI is required for methylation inhibition at the GATC1028 site; however, neither papI nor papB is required for inhibition of methylation at the GATC1130 site. We have identified a chromosomal locus, mbf (methylation-blocking factor), that is required for methylation protection of both the pap GATC1028 and GATC1130 sites. The mbf locus was identified after transposon mTn10 mutagenesis and mapped to 19.6 min on the E. coli chromosome. The effect of transposon mutations within mbf on pap pilin transcription was determined by using a papBAp-lac operon fusion which places lacZ under control of the papBA promoter. E. coli containing mbf::mTn10 and phase-off mbf+ E. coli cells both expressed beta-galactosidase levels about 30-fold lower than the beta-galactosidase level measured for phase-on mbf+ E. coli cells. These results indicated that mbf was necessary for pap pilin transcription and were supported by Northern (RNA) blotting and primer extension analyses. Moreover, transposon insertion within mbf greatly reduced Pap pilus expression. The mbf locus was isolated on a low-copy-number cosmid, pMBF1. Complementation analysis indicated that each of seven mbf::mTn10 mutants isolated contained a transposon insertion within the same gene or operon. The identification of the mbf locus, required for pap transcription, supports the hypothesis that pap phase variation is controlled by a mechanism involving alternation between different methylation states.

Amino Acid Sequence↗
PubMed1990

Regulation of pap pilin phase variation by a mechanism involving differential dam methylation states.

Transcription of the pap pilin (papA) gene in Escherichia coli is subject to control by a heritable phase variation mechanism in which alternation between transcriptionally active (phase on) and inactive (phase off) states occurs. Our results suggest that phase switching occurs without DNA rearrangement of pap DNA sequences, distinguishing this system from those described for E. coli type 1 pili and Salmonella flagellar phase variation. Analysis of the regulatory region upstream of papA in DNAs isolated from phase off and phase on cell populations showed that two deoxyadenosine methylase (Dam) sites, GATC1028 and GATC1130, were present. Southern blot analysis of MboI and DpnI restriction digests of DNAs showed that the GATC1028 site was unmethylated only in DNA isolated from phase on populations. Conversely, GATC1130 sites were unmethylated in DNA isolated from phase off populations. The presence of unmethylated GATC sites in E. coli is unusual and to our knowledge has not been previously reported. These results suggest that the methylation states of GATC1028 and GATC1130 may regulate pap transcription. Consistent with this hypothesis, Dam methylase levels affected the regulation of pap transcription; papA transcription was absent in dam- E. coli. Moreover, transition from the phase off to phase on state was not observed in E. coli expressing aberrantly high levels of Dam. A basic model is presented which outlines a possible mechanism by which alternation between phase off and phase on methylation states could occur.

Amino Acid Sequence↗
PubMed1990

Identification of an Escherichia coli genetic locus involved in thermoregulation of the pap operon.

We previously showed, using a single-copy papBAp-lac fusion (previously designated papBA-lac), that pyelonephritis-associated pili (pap) pilin gene transcription is subject to both phase variation and thermoregulatory control mechanisms (L. B. Blyn, B. A. Braaten, C. A. White-Ziegler, D. H. Rolfson, and D. A. Low, EMBO J. 8:613-620, 1989). At 37 degrees C, Escherichia coli strains carrying the papBAp-lac fusion displayed both Lac+ and Lac- colony phenotypes. In contrast, at 23 degrees C, colonies displayed a uniform Lac- phenotype, suggesting that pilin was not transcribed at this temperature. In this study, a strain carrying the papBAp-lac fusion was subjected to mini-Tn10 (mTn10) mutagenesis to isolate mutants that could initiate transcription of pilin at the nonpermissive temperature. Two classes of thermoregulatory mutants were identified in which the mTn10 mutation was linked to the mutant phenotype. Class I mutants displayed a phase variation phenotype at both 37 degrees C and 23 degrees C, whereas class II mutants displayed a uniform Lac+ colony phenotype at both temperatures. Preliminary analysis of these mutants showed that the mTn10 insertions in the class I mutants were chromosomally located, whereas the mTn10 insertions in the class II mutants were located within the papBAp-lac fusion phage. Southern blot analysis of the class I mutants demonstrated that mTn10 was present in the same 5.9-kilobase SalI DNA fragment in each mutant. Two of the class I mTn10 mutations were mapped to approximately 23.4 min on the E. coli K-12 chromosome. The locus defined by the class I mTn10 mutations was designated tcp, for thermoregulatory control of pap. Analysis of phase transition rates of the class I mutants showed that the phase-off (Lac-)----phase-on (Lac+) transition rates were higher than those observed with the nonmutant E. coli strain.

Blotting, Southern↗
PubMed1989

Phase-variation of pyelonephritis-associated pili in Escherichia coli: evidence for transcriptional regulation.

The regulation of pyelonephritis-associated pili (pap) pilin gene transcription has been examined using two operons (pap-17 and pap-21) isolated from the pyelonephritogenic Escherichia coli strain C1212. DNA sequence analysis and E. coli minicell analysis were used to map two genes (papB and papI) within the pilin regulatory regions of both pap-17 and pap-21, and the protein products of these genes were identified. Pilin transcription, initiated at the papBA promoter, was monitored by constructing single copy operon fusions with lacZYA in E. coli K-12. Inoculation of E. coli (pap'-lac) strains onto solid M9 minimal medium containing glycerol and the Lac indicator X-gal (M9-Glycerol) yielded both Lac+ and Lac- colony phenotypes. The Lac+ ("phase on') and Lac- ("phase off') phenotypes were heritable since reinoculation of M9-Glycerol with bacteria picked from Lac+ colonies gave rise to a much higher fraction of Lac+ colonies than reinoculation of M9-Glycerol with bacteria picked from Lac- colonies. Measurement of phase transition rates for E. coli (pap17'-lac) inoculated onto M9-Glycerol showed that the Lac(-)----Lac+ transition frequency (1.57 X 10(-4)/cell/generation) was reduced 35-fold when cells were inoculated onto minimal medium containing glucose (M9-Glucose). However, the Lac+----Lac-transition frequency obtained using M9-Glycerol (2.60 X 10(-2)/cell/generation) was 1.4-fold lower compared to results obtained with M9-Glucose. In contrast, lowering the incubation temperature of E. coli (pap17'-lac) cultures from 37 degrees C to 23 degrees C caused all cells to shift to the Lac- state.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1988

Isolation and comparison of Escherichia coli strains from canine and human patients with urinary tract infections.

We analyzed Escherichia coli strains isolated from dogs with urinary tract infections (UTIs) in an attempt to determine if any of these strains were similar to E. coli isolated from humans with UTIs. Using genotypic and phenotypic traits, we identified four canine and six human E. coli UTI isolates that all appeared to be closely related or identical. All isolates shared similar DNA sequences for pyelonephritis-associated pili (pap), alpha-hemolysin (hly), and insertion sequence 5 (IS5), on the basis of Southern blot analysis. Similar outer membrane protein, pilin, and plasmid profiles were obtained for each of the isolates, although minor heterogeneity was observed. All of these isolates expressed a neuraminidase-sensitive binding phenotype in contrast to the majority of human isolates, which are known to express an adhesin that recognizes terminal digalactoside residues. Taken together, these results suggest that similar E. coli uropathogens may be capable of infecting both dogs and humans. To determine if the intestinal tracts of dogs were a reservoir for uropathogenic E. coli, eight paired rectal and urine pap+ E. coli strains were cultured from dogs with UTIs. By using the same genotypic and phenotypic criteria described above as a basis for strain identity, seven of eight urine-rectal pairs showed intrapair identity. However, each urine-rectal pair displayed a unique overall profile and could be distinguished from the other pairs. We conclude that the uropathogen colonizing the bladders of dog can also be the predominant strain colonizing the intestinal tracts.

Animals↗
PubMed1982

Cells regulate their mitogenic response to thrombin through release of protease nexin.

We previously reported that human and mouse fibroblast-like cells release into their growth medium a protein that we termed protease nexin. Protease nexin forms a covalent acyl linkage with thrombin and certain other serine proteases via the protease active site and mediates their binding, internalization and degradation by cells. Binding of thrombin-protease nexin to cells is mediated by the protease nexin portion of the complex to a high-affinity cellular binding site. As thrombin is a potent mitogen for a variety of fibroblast-like cells in culture, we examined whether protease nexin itself regulates thrombin-stimulated cell division. Recently, we showed that heparin virtually blocked the binding of thrombin-protease nexin complexes to both mouse and human cells without affecting the ability of these cells to respond to thrombin. Thus, protease nexin does not appear to be a positive modulator in thrombin-induced cell division. Here, we show that protease nexin negatively regulates the mitogenic response of cells in culture to thrombin.

Amyloid beta-Protein Precursor↗