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D M Prescott

Publications and source records attributed to D M Prescott.

At least 37 records · Page 2Linked to original sources
PubMed1996

The germline gene encoding DNA polymerase alpha in the hypotrichous ciliate Oxytricha nova is extremely scrambled.

We report the structure of the micronuclear (germline) gene encoding the large catalytic subunit of DNA polymerase alpha (DNA pol alpha) in the ciliate Oxytricha nova. It contains 44 internal eliminated segments (IESs) that divide the gene into 45 macronuclear-destined segments (MDSs) that are in a non-randomly scrambled order with an inversion near the gene center. Odd numbered MDSs 29-43, containing 230 bp out of a total of 4938 bp of macronuclear sequence, are missing from the 14 kb cloned gene. The missing MDSs have not been located but are at least several kilobases from the main body of the gene. The remarkably scrambled DNA pol alpha gene must be extensively cut, re-ordered and spliced and an inversion must occur to produce an unscrambled, functional version of the gene during development of a new macronucleus. Unscrambling is hypothesized to occur by a homologous recombination mechanism guided by repeat sequences at MDS ends.

Animals↗
PubMed1996

Radiation plus local hyperthermia versus radiation plus the combination of local and whole-body hyperthermia in canine sarcomas.

PURPOSE: The purpose of this study was to assess the effect of increasing intratumoral temperatures by the combination of local hyperthermia (LH) and whole body hyperthermia (WBH) on the radiation response of canine sarcomas. METHODS AND MATERIALS: Dogs with spontaneous soft tissue sarcomas and no evidence of metastasis were randomized to be treated with radiation combined with either LH alone or LH + WBH. Dogs were accessioned for treatment at two institutions. The radiation dose was 56.25 Gy, given in 25 2.25 Gy daily fractions. Two hyperthermia treatments were given; one during the first and one during the last week of treatment. Dogs were evaluated after treatment for local recurrence, metastasis, and complications. RESULTS: Sixty-four dogs were treated between 1989 and 1993. The use of LH+WBH resulted in statistically significant increases in the low and middle regions of the temperature distributions. The largest increase was in the low temperatures with median CEM 43 T90 values of 4 vs. 49 min for LH vs. LH + WBH, respectively (p<0.001). There was no difference in duration of local tumor control between hyperthermia groups (p = 0.59). The time to metastasis was shorter for dogs receiving LH + WBH (p = 0.02); the hazard ratio for metastatic disease for dogs in the LH + WBH group was 2.4 (95% confidence interval, 1.2-5.4) with respect to dogs in the LH group. Complications were greater in larger tumors and in tumors treated with LH + WBH, CONCLUSION: The combination of LH + WBH with radiation therapy, as described herein, was not associated with an increase in local tumor control in comparison to use of LH with radiation therapy. The combination of LH + WBH also appeared to alter the biology of the metastatic process and was associated with more complications than LH. We identified no rationale for further study of LH + WBH in combination with radiation for treatment of solid tumors.

Animals↗
PubMed1996

A gene-sized DNA molecule encoding heat-shock protein 70 in Oxytricha nova.

The gene-sized DNA molecule in the macronucleus (mac) of Oxytricha nova (On) encoding heat-shock protein 70 (Hsp70) was cloned and sequenced. It contains 2654 bp, including telomeres. It consists of a 394-bp A+T-rich 5' leader, a 1956-bp open reading frame (ORF) encoding a putative polypeptide of 651 amino acids (aa), and a 240-bp A+T-rich 3' trailer. The 5' leader contains two copies of the 13-bp heat-shock element (HSE) consensus sequence of other eukaryotes. On responds to heat and CdCl2 stress with a major increase in hsp70 transcripts. The presence of HSE in the nucleotide sequence of hsp70 and the stress-induced in hsp70 transcripts are the first evidence of conventional transcriptional regulation of a hypotrich gene-size DNA molecule. The ORF has large putative ATPase and polypeptide-binding domains with high aa identity to Hsp70 polypeptides of other eukaryotes.

Amino Acid Sequence↗
PubMed1996

1H MRI phase thermometry in vivo in canine brain, muscle, and tumor tissue.

The temperature sensitivity of the chemical shift of water (approximately 0.01 ppm/degree C) provides a potential method to monitor temperature changes in vivo or in vitro through the changes in phase of a gradient-echo magnetic resonance (MR) image. This relation was studied at 1.5 T in gel materials and in vivo in canine brain and muscle tissue, heated with a radio frequency (rf) annular phased array hyperthermia antenna. The rf fields associated with heating (130 MHz) and imaging (64 MHz) were decoupled using bandpass filters providing isolation in excess of 100 dB, thus allowing simultaneous imaging and rf heating without deterioration of the MR image signal-to-noise ratio. In a gel, temperature sensitivity of the MR image phase was observed to be (4.41 +/- 0.02) phase degrees/degree C for Te = 20 ms, which allowed temperature changes of 0.22 degree C to be resolved for a 50-mm3 region in less than 10 s of data acquisition. In vivo, for Te = 20 ms, the temperature sensitivity was (3.2 +/- 0.1) phase degrees/degree C for brain tissue, (3.1 +/- 0.1) phase degrees/degree C for muscle, and (3.0 +/- 0.2) phase degrees/degree C for a muscle tumor (sarcoma), allowing temperature changes of 0.6 degree C to be resolved in a 16-mm3 volume in less than 10 s of data acquisition. We conclude that, while the technique is very sensitive to magnetic field inhomogeneity, stability, and subject motion, it appears to be useful for in vivo temperature change measurement.

Animals↗
PubMed1996

Monitoring of neoadjuvant therapy response of soft-tissue and musculoskeletal sarcoma using fluorine-18-FDG PET.

UNLABELLED: The purpose of this study was to investigate the potential role of FDG-PET in the monitoring of neoadjuvant therapy of soft-tissue and musculoskeletal sarcomas. METHODS: Nine patients were studied. Neoadjuvant therapy consisted of either chemotherapy or combined radiotherapy and hyperthermia. The FDG-PET studies were obtained, when possible, prior to therapy, 1-3 wk after commencement of therapy, and prior to surgery after completion of neoadjuvant therapy. In two patients, all three studies were completed. The remainder of patients underwent one or two studies at varying timepoints. RESULTS: In tumors treated with combined radiotherapy and hyperthermia, well-defined regions of absent uptake developed within responsive tumors, correlating pathologically with necrosis. Following treatment, a peripheral rim of FDG accumulation was found to correlate pathologically with the formation of a fibrous pseudocapsule. In tumors treated with chemotherapy, FDG accumulation decreased more homogeneously throughout the tumor, in responsive cases. Despite 100% tumor cell kill in some patients, persistent tumor FDG uptake was observed which correlated pathologically with uptake within benign therapy-related fibrous tissue. Significant FDG accumulation was also observed at the site of an uncontaminated incisional biopsy. CONCLUSION: These initial results demonstrate changes in tumor accumulation of FDG during and after neoadjuvant therapy; these changes are dependent on the type of neoadjuvant therapy administered. Prominent FDG accumulation was observed in benign tissues both within and adjacent to the treated tumor.

Adolescent↗
PubMed1995

Phosphorylation of the Oxytricha telomere protein: possible cell cycle regulation.

In the macronucleus of the ciliate Oxytricha nova, telomeres end with single-stranded (T4G4)2 DNA bound to a heterodimeric telomere protein (alpha beta). Both the alpha and beta subunits (alpha-TP and beta-TP) were phosphorylated in asynchronously growing Oxytricha; beta-TP was phosphorylated to a much higher degree. In vitro, mouse cyclin-dependent kinases (Cdks) phosphorylated beta-TP in a lysine-rich domain that is not required for specific DNA binding but is implicated in higher order structure formation of telomeres. Therefore, phosphorylation of beta-TP could modulate a function of the telomere protein that is separate from specific DNA binding. Phosphoamino acid analysis revealed that the mouse Cdks modify predominantly threonine residues in beta-TP, consistent with the observation that beta-TP contains two consensus Cdk recognition sequences containing threonine residues. In Xenopus egg extracts that undergo cell cycling, beta-TP was phosphorylated in M phase and dephosphorylated in interphase. This work provides the first direct evidence of phosphorylation at telomeres in any organism, as well as indirect evidence for cell cycle regulation of telomere phosphorylation. The Cdc2/cyclin A and Cdc2/cyclin B kinases are required for major mitotic events. An attractive model is that phosphorylation of beta-TP by these kinases is required for the breakdown of telomere associations with each other and/or with nuclear structures prior to nuclear division.

Amino Acid Sequence↗
PubMed1995

Scrambling of the actin I gene in two Oxytricha species.

The DNA in a germ-line nucleus (a micronucleus) undergoes extensive processing when it develops into a somatic nucleus (a macronucleus) after cell mating in hypotrichous ciliates. Processing includes destruction of a large amount of spacer DNA between genes and excision of gene-sized molecules from chromosomes. Before processing, micronuclear genes are interrupted by numerous noncoding segments called internal eliminated sequences (IESs). The IESs are excised and destroyed, and the retained macro-nuclear-destined sequences (MDSs) are spliced. MDSs in some micronuclear genes are not in proper order and must be reordered during processing to create functional gene-sized molecules for the macronucleus. Here we report that the micronuclear actin I gene in Oxytricha trifallax WR consists of 10 MDSs and 9 IESs compared to the previously reported 9 MDSs and 8 IESs in the micronuclear actin I gene of Oxytricha nova. The MDSs in the actin I gene are scrambled in a similar pattern in the two species, but the positions of MDS-IES junctions are shifted by up to 14 bp for scrambled and 138 bp for the nonscrambled MDSs. The shifts in MDS-IES junctions create differences in the repeat sequences that are believed to guide MDS splicing. Also, the sizes and sequences of IESs in the micronuclear actin I genes are different in the two Oxytricha species. These observations give insight about the possible origins of IES insertion and MDS scrambling in evolution and show the extraordinary malleability of the germ-line DNA in hypotrichs.

Actins↗
PubMed1995

Macronuclear gene-sized molecules of hypotrichs.

The macronuclear genome of hypotrichous ciliates consists of DNA molecules of gene-sized length. A macronuclear DNA molecule contains a single coding region. We have analyzed the many hypotrich macronuclear DNA sequences sequenced by us and others. No highly conserved promoter sequences nor replication initiation sequences have been identified in the 5' nor in the 3' non-translated regions, suggesting that promoter function in hypotrichs may differ from other eukaryotes. The macronuclear genes are intron-poor; approximately 19% of the genes sequenced to date have one to three introns. Not all macronuclear DNA molecules may be transcribed; some macronuclear molecules may not have any coding function. Codon bias in hypotrichs is different in many respects from other ciliates and from other eukaryotes.

Animals↗
PubMed1994

Changes in plasma TGF beta levels during pulmonary radiotherapy as a predictor of the risk of developing radiation pneumonitis.

PURPOSE: To determine whether plasma transforming growth factor-beta (TGF-beta) levels measured before and during radical radiotherapy for lung cancer could be used to predict patients at risk for the development of radiation pneumonitis. METHODS AND MATERIALS: The first eight patients with lung cancer (nonsmall cell: seven, small cell: one) enrolled in a prospective study designed to evaluate physiological and molecular biologic correlates of radiation induced normal tissue injury are described. The study began in June 1991. All patients were treated with radiotherapy with curative intent. Plasma transforming growth factor-beta levels were obtained before, weekly during, and at each follow-up after treatment. Pretreatment pulmonary function tests and single photon emission computed tomography scans were obtained to assess baseline lung function and were repeated at follow-up visits. Dose-volume histogram analyses were performed to determine the volume of lung which received > or = 30 Gy. Patients were assessed at each follow-up visit for signs and symptoms of pneumonitis. RESULTS: Five patients developed signs and/or symptoms of pulmonary injury consistent with pneumonitis and three patients did not. In all three patients not developing pneumonitis, plasma TGF-beta levels normalized by the end of radiotherapy. In contrast, four out of five patients who suffered pneumonitis had persistently elevated plasma TGF-beta levels by the end of therapy. This finding appeared to be independent of the volume of irradiated lung. CONCLUSIONS: These results suggest that plasma TGF-beta levels during treatment may be useful to determine which patients are at high risk of developing symptomatic pneumonitis following thoracic radiotherapy. This finding may have implications when planning additional therapy (either chemotherapy or radiotherapy) which may have potentially adverse consequences on the lung.

Adult↗
PubMed1994

A gene-sized DNA molecule encoding the catalytic subunit of DNA polymerase alpha in the macronucleus of Oxytricha nova.

We have isolated a gene-sized molecule encoding the catalytic subunit of DNA polymerase alpha from a macronuclear genomic library of Oxytricha nova, by using a 0.7-kb fragment of the corresponding human gene as a hybridization probe. Two different versions of the gene are present in the macronucleus, one with an EcoRI site (RI+) and one without an EcoRI site (RI-). The cloned RI- version has been characterized. It is 4938 bp in length, excluding telomeres. It consists of a 329-bp 5' leader, a 4479-bp coding region and a 130-bp 3' trailer. The deduced amino-acid sequence shares conserved regions with the yeast and human polypeptides. We also demonstrate by Southern analysis that gene-sized molecules of similar size, homologous to the isolated O. nova gene are present in the mac genome of closely and distantly related hypotrichs.

Animals↗
PubMed1994

The actin II-encoding gene in the macronucleus of Oxytricha nova.

The macronucleus of the hypotrichous ciliate Oxytricha nova contains two DNA molecules with sizes of approx. 1.4 (actin II) and approx. 1.6 (actin I) kb that encode actin (Act) proteins. The 1.6-kb molecule has been previously described [Greslin et al., DNA 7 (1988) 529-536]. The 1.4-kb molecule is described here. The cloned molecule is 1393 bp in length, including 20 bp of telomere sequence at each end. It has an open reading frame (ORF) of 1119 bp that encodes a putative polypeptide of 374 amino acids (aa) designated as ActII. No introns are present. The coding segment is preceded at its 5' end by an A + T-rich 5' noncoding segment of 100 bp, excluding the telomere, and is followed by an A + T-rich 3' noncoding segment of 124 bp, also excluding the telomere. The aa sequence of the actII gene is 63% identical to actI of O. nova and 68, 70 and 69% identical to the rabbit, yeast and Tetrahymena Act aa sequences, respectively. ActI in O. nova is 64 to 68% identical to Act in other eukaryotes. Thus, the two Act in O. nova differ substantially from one another and neither conforms to the conserved Act sequence generally present in eukaryotes.

Actins↗
PubMed1994

Therapy monitoring in human and canine soft tissue sarcomas using magnetic resonance imaging and spectroscopy.

PURPOSE: The goals of this study were to determine whether magnetic resonance parameters (a) can identify early during therapy those patients most likely to respond to hyperthermia and radiotherapy, (b) can provide prior to or early during therapy information about the temperature distributions which can be obtained in patients receiving hyperthermia, and (c) can provide an understanding of the effects of hyperthermia on tumor metabolic status. METHODS AND MATERIALS: Twenty-one human patients and 10 canine patients with soft tissue sarcomas treated with preoperative hyperthermia and radiation had a series of magnetic resonance imaging and phosphorous spectroscopy studies done. To address the goals for both the human and canine populations, changes in mean T2 relaxation times, pH, and various phosphometabolite ratios from the pretreatment (Study 1) to the post first hyperthermia study (Study 2) were correlated with treatment outcome; pretreatment magnetic resonance parameters and changes in magnetic resonance parameters (Study 2-Study 1) were compared with various cumulative thermal descriptors; and thermal descriptors of the first hyperthermia were compared with changes in magnetic resonance phosphometabolite ratios. RESULTS: A decrease in adenosine triphosphate/phosphomonoester from study 1 to study 2 is associated with a greater chance of > or = 95% necrosis in surgical resected tumors from human patients, but no significant relationships were observed between changes in tumor pH or phosphometabolite ratios and time to local failure in dogs. Pretreatment magnetic resonance parameters correlated with various thermal dose descriptors in canines but not in humans. Change in adenosine triphosphate/inorganic phosphate and phosphomonoester signal to noise ratio correlated with cumulative thermal descriptors in dogs and humans, respectively. In dogs only, increases in thermal dose resulted in decreases in high energy phosphometabolites. CONCLUSION: Changes in magnetic resonance parameters early during therapy may be predictive of treatment outcome. Pretreatment and changes in magnetic resonance parameters appear to predict how well a tumor will be heated during hyperthermia. Magnetic resonance spectroscopy also appears to be a useful tool to study the effects of various thermal doses on tumor metabolic status.

Adenosine Triphosphate↗
PubMed1994

The DNA of ciliated protozoa.

Ciliates contain two types of nuclei: a micronucleus and a macronucleus. The micronucleus serves as the germ line nucleus but does not express its genes. The macronucleus provides the nuclear RNA for vegetative growth. Mating cells exchange haploid micronuclei, and a new macronucleus develops from a new diploid micronucleus. The old macronucleus is destroyed. This conversion consists of amplification, elimination, fragmentation, and splicing of DNA sequences on a massive scale. Fragmentation produces subchromosomal molecules in Tetrahymena and Paramecium cells and much smaller, gene-sized molecules in hypotrichous ciliates to which telomere sequences are added. These molecules are then amplified, some to higher copy numbers than others. rDNA is differentially amplified to thousands of copies per macronucleus. Eliminated sequences include transposonlike elements and sequences called internal eliminated sequences that interrupt gene coding regions in the micronuclear genome. Some, perhaps all, of these are excised as circular molecules and destroyed. In at least some hypotrichs, segments of some micronuclear genes are scrambled in a nonfunctional order and are recorded during macronuclear development. Vegetatively growing ciliates appear to possess a mechanism for adjusting copy numbers of individual genes, which corrects gene imbalances resulting from random distribution of DNA molecules during amitosis of the macronucleus. Other distinctive features of ciliate DNA include an altered use of the conventional stop codons.

Animals↗
PubMed1994

MR imaging and spectroscopy for prognostic evaluation in soft-tissue sarcomas.

PURPOSE: To enable prediction of tumor response to a particular treatment. MATERIALS AND METHODS: The authors studied the value of hydrogen-1 T2 and phosphorus-31 magnetic resonance (MR) spectroscopic metabolic determinations as indicators of prognosis in 20 humans and 10 dogs with soft-tissue sarcomas. All patients underwent combined fractionated radiation therapy and hyperthermia. Surgical resection of the entire tumor in humans allowed comparison of pretherapy T2 and MR spectroscopic parameters with subsequent histologic findings. Long-term clinical follow-up (no surgical intervention) was available with the dogs and allowed comparison of pretherapy pH with the duration of local relapse-free survival. RESULTS: A relationship exists between pretherapy pH and T2 and ultimate tumor necrosis in humans and pretherapy pH and time elapsed until local failure in dogs. CONCLUSION: MR imaging and spectroscopy could be useful in the prognosis of patients with soft-tissue sarcomas before therapy is initiated.

Adult↗
PubMed1993

Restructuring of DNA sequences in the germline genome of Oxytricha.

Most genes in the germline genome of hypotrichous ciliates are crippled by the presence of interrupting sequences. Some genes are additionally impaired because their sequences are in disorder. These gene defects are corrected when germline chromosomal DNA sequences are amplified, cut, spliced, reordered, and eliminated to produce somatic DNA.

Animals↗
PubMed1992

Cutting, splicing, reordering, and elimination of DNA sequences in hypotrichous ciliates.

Hypotrichous ciliates extensively process genomic DNA during their life cycle. Processing occurs after cell mating, beginning with multiple rounds of DNA replication to form polytene chromosomes. Thousands of transposonlike elements are then excised from the chromosomes and destroyed, and thousands of short, internal eliminated sequences (IESs) are excised from coding and noncoding parts of genes and destroyed. IES removal from a gene is accompanied by splicing of the remaining chromosomal DNA segments to form a transcriptionally competent gene. For some genes these DNA segments are in a scrambled order and are ligated into a genetically correct order at the time of IES removal. Next the polytene chromosomes are cut up band-by-band and all genes are excised from the chromosomes as short, linear molecules averaging 2.2 kbp (in Oxytricha nova). Gene excision is accompanied by destruction of all nongenic DNA, which, together with the transposonlike elements and IESs, accounts for approximately 95% of the total sequence complexity of the genome in O. nova. Telomeric sequences are added to the excised gene-sized DNA molecules. Finally, the gene-sized molecules are replicated several times to form the macronucleus of the organism.

Actins↗
PubMed1992

Scrambled actin I gene in the micronucleus of Oxytricha nova.

In the hypotrichous ciliate Oxytricha nova the cloned precursor gene from the micronuclear genome that encodes actin I is composed of highly disordered blocks of deoxynucleotide sequences. We present and illustrate in detail a recombination model that explains how the actin I gene may be unscrambled during macronuclear development after cell mating. The model was described in a previous publication (Greslin et al.: Proc Natl Acad Sci USA 86:6264-6268, 1989). Here we show the data, described in the earlier publication, that support the model. The data show that scrambling is not an artifact of cloning. They rule against the presence of an unscrambled copy of the actin I gene in the micronucleus, which means that unscrambling must be a part of macronuclear development. Finally, the data prove that the actin I gene in O. trifallax is scrambled in a pattern that resembles the pattern in O. nova.

Actins↗