The properties of a tRNA-specific adenosine deaminase from Drosophila melanogaster support an evolutionary link between pre-mRNA editing and tRNA modification.

Pre-mRNA editing involving the conversion of adenosine to inosine is mediated by adenosine deaminases that act on RNA (ADAR1 and ADAR2). ADARs contain multiple double-stranded RNA(dsRNA)-binding domains in addition to an adenosine deaminase domain. An adenosine deaminase acting on tRNAs, scTad1p (also known as scADAT1), cloned from Saccharomyces cerevisiae has a deaminase domain related to the ADARs but lacks dsRNA-binding domains. We have identified a gene homologous to scADAT1 in the region of Drosophila melanogaster Adh chromosome II. Recombinant Drosophila ADAT1 (dADAT1) has been expressed in the yeast Pichia pastoris and purified. The enzyme has no activity on dsRNA substrates but is a tRNA deaminase with specificity for adenosine 37 of insect alanine tRNA. dADAT1 shows greater similarity to vertebrate ADARs than to yeast Tad1p, supporting the hypothesis of a common evolutionary origin for ADARs and ADATs. dAdat1 transcripts are maternally supplied in the egg. Zygotic expression is widespread initially and later concentrates in the central nervous system.

Image segmentation of trabecular spongiosa by visual inspection of the gradient magnitude.

Recent advances in physical models of skeletal dosimetry utilize high-resolution 3-dimensional microscopic computed tomography images of trabecular spongiosa. These images are coupled to radiation transport codes to assess energy deposition within active bone marrow and trabecular endosteum. These transport codes rely primarily on the segmentation of the spongiosa images into bone and marrow voxels. Image thresholding has been the segmentation of choice for bone sample images because of its extreme simplicity. However, the ability of the segmentation to reproduce the physical boundary between bone and marrow depends on the selection of the threshold value. Statistical models, as well as visual inspection of the image, have been employed extensively to determine the correct threshold. Both techniques are affected by partial volume effect and can provide unexpected results if performed without care. In this study, we propose a new technique to threshold trabecular spongiosa images based on visual inspection of the image gradient magnitude. We first show that the gradient magnitude of the image reaches a maximum along a surface that remains almost independent of partial volume effect and that is a good representation of the physical boundary between bone and marrow. A computer program was then developed to allow a user to compare the position of the iso-surface produced by a threshold with the gradient magnitude. The threshold that produces the iso-surface that best coincides with the maximum gradient is chosen. The technique was finally tested with a set of images of a true bone sample with different resolutions, as well as with three images of a cube of Duocell aluminium foam of known mass and density. Both tests demonstrate the ability of the gradient magnitude technique to retrieve sample volumes or media volume fractions with 1% accuracy at 30 microm voxel size.

Progress report of combined chemoradiotherapy versus radiotherapy alone in patients with esophageal cancer: an intergroup study.

PURPOSE: The present intergroup phase III randomized study compared combined chemotherapy (CT) plus radiotherapy (RT) treatment versus RT only in patients with locally advanced esophageal cancer. MATERIALS AND METHODS: Two courses of chemotherapy during 50 Gy RT followed by additional two courses of the same CT, versus 64 Gy RT alone were investigated. CT consisted of cisplatin 75 mg/m2 on day 1 [corrected] and fluorouracil (5FU) 1,000 mg/m2/d on days 1 to 4 every 4 weeks with RT and every 3 weeks post-RT. The main objective of the study was to compare overall survival between the two randomized treatment groups. Patients were stratified by tumor size, histology, and degree of weight loss. RESULTS: Sixty-two assessable patients were randomized to receive RT alone, and 61 to the combined arm. Patients characteristics were as follows: squamous cell cancer, 90% versus 85%; weight loss greater than 10 lb, 61% versus 69%; and tumor size, > or = 5 cm, 82% versus 80% on the RT and CT-RT arms, respectively. Systemic side effects, which consisted of nausea, vomiting, and renal and myelosuppression, occurred more frequently on the combined arm, while local side effects were similar in both groups. With a minimum follow-up time of 5 years for all patients, the median survival duration was 14.1 months and the 5-year survival rate was 27% in the combined treatment group, while the median survival duration was 9.3 months with no patients alive at 5 years in the RT-alone group (P < .0001). Additional patients (69) were treated with the same combined therapy and were analyzed. The results of the last group confirmed all of the results obtained with combined CT-RT in the randomized trial, with a median survival duration of 17.2 months and 3-year survival rate of 30%. CONCLUSION: We conclude that cisplatin and 5FU infusion given during and post-RT of 50 Gy is statistically superior to standard 64-Gy RT alone in patients with locally advanced esophageal cancer.

Pelvic lymphadenectomy combined with transperineal interstitial implantation of iridium-192 and external beam radiotherapy for locally advanced prostatic carcinoma: technical description.

Regardless of the treatment modality, control of locally advanced extracapsular prostatic cancer remains a therapeutic challenge. At the Mayo Clinic, we have recently developed a combined approach for surgically staged patients employing interstitial irradiation with 192 Iridium via a transperineal template. The Martinez Universal Perineal Interstitial Template (MUPIT-II) and moderate doses of external beam irradiation. The procedure consists of: 1) preoperative single dose external beam irradiation to decrease potential for tumor seeding or showering of malignant cells during MUPIT-II placement; 2) adequate surgical staging through a bilateral retroperitoneal lymphadenectomy; 3) transperineal interstitial implantation of the tumor and retropubic palpation of the needles to verify proper position; 4) intraoperative X rays are taken and used for documentation of needle position as well as for calculation and optimization of the implant dose distribution with the aid of a computerized system; 5) moderate doses of external beam irradiation postoperatively to the prostate with adequate margins. Our favorable preliminary results obtained in 18 patients treated with the above approach warrant the continuation of this trial. Details of this technique are presented.

Pelvic lymphadenectomy and transperineal interstitial implantation of IR 192 combined with external beam radiotherapy for bulky stage C prostatic carcinoma.

From January 1983 until June 1987, 51 patients with locally advanced prostatic carcinoma (47 Stage C, 4 bulky B2) were treated at Mayo Clinic (33 patients) and at William Beaumont Hospital (18 patients) with (a) 5 Gy delivered pre-operatively in one fraction, (b) pelvic lymphadenectomy and (c) interstitial implantation of the prostate with Ir 192 seeds via a perineal template (the Martinez Universal Perineal Interstitial Template) to deliver 35 Gy, and (d) 30.6 Gy external beam therapy in 17 fractions to prostate only fields. Initial clinical response has been excellent. Local control, with a median follow-up of 45 months, has been 100% by clinical exam and 84.5% pathologically in the subset biopsied. Disease-free actuarial survival at 5 years is 89%. Major toxicity has been limited to the rectum, but a modification of the brachytherapy technique has reduced this sharply. We conclude that bulky Stage C prostatic carcinoma can be successfully treated by this aggressive combination of modalities with acceptable toxicity.

Locally recurrent non-small-cell lung cancer after complete surgical resection.

Between Jan. 1, 1976, and Dec. 31, 1985, at our institution, 37 patients who had undergone prior complete surgical resection of non-small-cell lung cancer received definitive thoracic radiation therapy (TRT) for locally recurrent disease. Of the 37 recurrences, 33 were in the pulmonary parenchyma or the hilar, mediastinal, or supraclavicular lymph nodes; the other 4 were in the chest wall. The initial stage of disease was I in 43%, II in 35%, and IIIA in 19%, whereas at the time of local recurrence, the stage was I in 8%, II in 11%, IIIA in 57%, IIIB in 22%, and IV in 3% (this patient had multiple pulmonary nodules encompassible within a single TRT field). The locally recurrent lesions were squamous cell carcinoma in 30%, adenocarcinoma or large-cell carcinoma in 46%, mixed types in 5%, and unknown type in 19%. All patients received megavoltage TRT, most often 4,000 cGy in 10 fractions administered in a split-course schedule. In addition, 15 patients received multiagent chemotherapy, usually a combination of cyclophosphamide, doxorubicin hydrochloride, and cisplatin or a regimen that included these drugs. The 2-year and 5-year survivals were 30% and 4%, respectively, and the median duration of survival was 13.7 months. Survival was not improved by the addition of chemotherapy. Approximately half of the patients had radiographic and symptomatic responses after TRT. Of 33 patients assessable for post-TRT patterns of failure, 46% had local failure only, 18% had local plus systemic failure, and 32% had systemic failure only. Two-thirds of the patients died as a direct consequence of progressive chest disease, despite receiving TRT.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute toxicity and preliminary therapeutic results of pelvic lymphadenectomy combined with transperineal interstitial implantation of 192IR and external beam radiotherapy for locally advanced prostate cancer.

We have developed a combined modality approach for treatment of locally advanced prostate cancer consisting of: surgical staging with lymphadenectomy; transperineal placement of afterloading needles for a course of 192Ir brachytherapy; and a course of moderate-dose external beam irradiation to tight prostatic fields. The flexibility of the treatment permits adaptation of the dose to any tumor configuration. A total of 23 patients have been treated to date; 18 are available for treatment review and 14 for acute toxicity analysis. Significant morbidity has occurred in only 2 patients, 1 with a pelvic hematoma and 1 with hematoma in the wound. Local tumor response has been excellent in all but 1 patient. A good degree of symptomatic improvement was observed at the three-month follow-up. This approach appears to be safe and effective at this time. A brief review of the technique and the rationale for its use are also presented.

Protective effect of antioxidants on the impairment of sperm motility by activated polymorphonuclear leukocytes.

OBJECTIVE: To test the ability of antioxidants to reduce the loss of sperm motility caused by reactive oxygen species generated by polymorphonuclear leukocytes (PML). DESIGN: Standardized preparations of leukocyte-contaminated semen were created by suspending known concentrations of purified spermatozoa and PML in seminal plasma. After the stimulation of reactive oxygen species generation with phorbol ester, the spermatozoa were washed and incubated in culture medium before an analysis of their movement. The ability of antioxidants to counteract the free radical-induced loss of sperm motility observed under these circumstances was assessed. SETTING: An institutional research laboratory. PARTICIPANTS: The semen was obtained from normal volunteers. INTERVENTIONS: The following were tested: vitamins C and E, dimethylsulfoxide, catalase, hypotaurine, N-acetylcysteine, and reduced glutathione. MAIN OUTCOME MEASURES: Reactive oxygen species generation was monitored by luminol-dependent chemiluminescence. Sperm motility was assessed manually and by computer-aided semen analysis. RESULTS: Consistent impairment of sperm motility and average path velocity was observed in the presence of activated PML. This effect was reduced by the concomitant presence of glutathione, N-acetylcysteine, hypotaurine, and catalase. CONCLUSION: Antioxidants can protect against the damaging effect of leukocyte-derived reactive oxygen species on sperm movement and may be of clinical value in assisted conception procedures.

Development of an image analysis system to monitor the retention of residual cytoplasm by human spermatozoa: correlation with biochemical markers of the cytoplasmic space, oxidative stress, and sperm function.

A method has been developed for quantifying the residual cytoplasm present in the midpiece of human spermatozoa, based upon the imaging of NADH oxidoreductase activity. This procedure used NADH and nitroblue tetrazolium as electron donor and acceptor, respectively, and resulted in the discrete staining of the entire midpiece area, including the residual cytoplasm. Image analysis techniques were then used to generate binary images of the midpiece, from which objective measurements of this cellular domain could be undertaken. Such data were found to be highly correlated with biochemical markers of the cytoplasmic space, such as creatine kinase (CK) and glucose-6-phosphate dehydrogenase (G-6-PDH), in sperm populations depleted of detectable leukocyte contamination. Morphometric analysis of the sperm midpiece was also found to reflect semen quality in that it predicted the proportion of the ejaculate that would be recovered from the high-density region of Percoll gradients and was negatively correlated with the movement and morphology of the spermatozoa in semen. Variation in the retention of excess residual cytoplasm was also associated with differences in the functional competence of washed sperm preparations, both within and between ejaculates. Thus, within-ejaculate comparisons of high- and low-density sperm subpopulations revealed a relative disruption of sperm function in the low-density fraction. This disruption was associated with the presence of excess residual cytoplasm in the midpiece, high concentrations of cytoplasmic enzymes, and the enhanced-generation reactive oxygen species (ROS). Functional differences between individual high-density Percoll preparations were also negatively correlated with the area of the midpiece and the corresponding capacity of the spermatozoa to generate ROS. These findings suggest that one of the factors involved in the etiology of defective sperm function is the incomplete extrusion of germ cell cytoplasm during spermiogenesis as a consequence of which the spermatozoa experience a loss of function associated with the induction of oxidative stress.

A hyperboliod representation of the bone-marrow interface within 3D NMR images of trabecular bone: applications to skeletal dosimetry.

Recent advances in physical models of skeletal dosimetry utilize high-resolution NMR microscopy images of trabecular bone. These images are coupled to radiation transport codes to assess energy deposition within active bone marrow irradiated by bone- or marrow-incorporated radionuclides. Recent studies have demonstrated that the rectangular shape of image voxels is responsible for cross-region (bone-to-marrow) absorbed fraction errors of up to 50% for very low-energy electrons (<50 keV). In this study, a new hyperboloid adaptation of the marching cube (MC) image-visualization algorithm is implemented within 3D digital images of trabecular bone to better define the bone-marrow interface, and thus reduce voxel effects in the assessment of cross-region absorbed fractions. To test the method, a mathematical sample of trabecular bone was constructed, composed of a random distribution of spherical marrow cavities, and subsequently coupled to the EGSnrc radiation code to generate reference values for the energy deposition in marrow or bone. Next, digital images of the bone model were constructed over a range of simulated image resolutions, and coupled to EGSnrc using the hyperboloid MC (HMC) algorithm. For the radionuclides 33P, 117mSn, 131I and 153Sm, values of S(marrow<--bone) estimated using voxel models of trabecular bone were shown to have relative errors of 10%, 9%, <1% and <1% at a voxel size of 150 microm. At a voxel size of 60 microm, these errors were 6%, 5%, <1% and <1%, respectively. When the HMC model was applied during particle transport, the relative errors on S(marrow<--bone) for these same radionuclides were reduced to 7%, 6%, <1% and <1% at a voxel size of 150 microm, and to 2%, 2%, <1% and <1% at a voxel size of 60 microm. The technique was also applied to a real NMR image of human trabecular bone with a similar demonstration of reductions in dosimetry errors.

Final report of the general clinical results of the British Institute of Radiology fractionation study of 3F/wk versus 5F/wk in radiotherapy of carcinoma of the laryngo-pharynx.

The 10 year follow-up of a clinical trial involving the comparison of 3F/wk versus 5F/wk in radiotherapy of squamous cell carcinoma of the larynx and hypopharynx has now been completed. The trial involved an intake of 734 patients between 1966 and 1975. The classification of all patients has been revised to conform with the latest TNM publication. A reduction in total dose was made for 3F/wk compared with 5F/wk. This varied between 13% and 11% in centres treating over 3 weeks and 6 weeks, respectively. No statistically significant differences have been found between the two arms (3F/wk versus 5F/wk) of the trial in any of the main group analyses. A number of sub-group analyses relating to survival, tumour-free and laryngectomy-free rates and to the comparison of acute or late normal-tissue radiation damage have also been performed. No differences have been found that could be considered to be statistically significant in relation to the particular sub-group. Previous interim reports suggested minor differences in sub-group analyses between the 3F/wk and 5F/wk regimes in this trial; these have diminished now that the full follow-up data are available. This trial has provided evidence on which clinicians may base their choice between either a 3F/wk fractionation regime or a conventional 5F/wk treatment protocol in the treatment of carcinoma of the laryngo-pharynx.

First interim progress report on the second British Institute of Radiology Fractionation Study: short versus long overall treatment times for radiotherapy of carcinoma of the laryngopharynx.

Preliminary data from a second British Institute of Radiology Fractionation Trial comparing short (less than or equal to 4 weeks) and long (greater than 4 weeks) overall treatment times are reported. The intake of patients ran from January, 1975 to December, 1985 when 611 patients with carcinoma of the laryngo-pharynx were registered in this prospective, randomized, multicentre clinical trial. No significant differences have, so far, been demonstrated between the two arms of the trial with respect to observed survival rates, tumour-free or laryngectomy-free rates. Further long-term follow-up is continuing.

Final report on the second British Institute of Radiology fractionation study: short versus long overall treatment times for radiotherapy of carcinoma of the laryngo-pharynx.

The second British Institute of Radiology trial of dose fractionation in radiotherapy compared two groups of prospectively randomized patients with squamous carcinoma of the laryngo-pharynx; one group was treated in a short (less than or equal to 4 weeks) and the other in a long (greater than 4 weeks) overall time. Treatment in any one centre could be given, with no planned gap in the course of treatment, either as a conventional, daily (5 fractions per week regime) or as 3 fractions per week. A total of 611 patients were allocated to treatment, of whom nine have had to be excluded from the analysis for a lack of information. Patients were admitted to the trial from January 1976 to December 1985 and were followed up for a maximum of 10 years and a minimum of 3 years. A reduction in total dose was made for use in the short compared with the long treatment regime. This reduction in total dose varied between 18% and 22% depending on whether 5 fractions or 3 fractions per week regimes were used. Overall, no statistically significant differences have been found between the two arms of the trial. The patients treated with 5 fractions per week in a short overall treatment time showed fewer late normal tissue effects. An analysis based on stratification by age, stage and anatomical site gave a relative risk (short/long overall treatment time) for deaths of 1.23 with a 95% confidence interval from 0.96 to 1.59. Analyses stratified for stage and site gave relative risks with 95% confidence intervals of 1 x 10 (0.84-1.44) for local recurrences/tumour persistence, and 1.01 (0.70-1.45) for laryngectomies.

Sixth interim progress report of the British Institute of Radiology fractionation study of 3F/week versus 5F/week in radiotherapy of the laryngo-pharynx.

The results are reported of the multicentre BIR fractionation trial of 3F/week versus 5F/week in radiotherapy of the laryngo-pharynx. 687 patient records have been analysed with respect to survival rates, recurrence-free rates and laryngectomy-free rates. For the group as a whole these analyses show no difference between the two fractionation regimes. Analysis of the sub-group which had early disease confined to the vocal cords does, however, show a better recurrence-free and laryngectomy-free rate for those patients treated with 5F/week, though the survival rate for the two groups remains similar. Acute and late normal tissue reactions are reported for up to six years after treatment. It appears that treatment with 3F/week can be given safely to patients with advanced disease. The differences between the two treatment groups who had early disease of the vocal cords are discussed, but until more data become available in the future the problems raised cannot be resolved.

Seventh interim progress report of the british institute of radiology fractionation study of 3F/week versus 5F/week in radiotherapy of the laryngo-pharynx.

A fresh analysis of the data entered into the multicentre BIR fractionation trial of 3F/week versus 5F/week in radiotherapy of the laryngopharynx has been undertaken. Completed records of the 732 patients initially entered into the trial have now risen from 687 at the last report to 706. The data have been analysed in a manner similar to that adopted previously so as to measure the effects of the two regimes on both tumour and normal tissues, and some additional analyses have now also been made. There have been some modifications in the results in the various sub-groups which may be due to an inadequate number of patients having been followed up for long enough at the time of the previous analyses. More data for late radiation damage to normal tissues and new radiobiological findings have suggested possible explanations for the differences which have emerged between the two groups. The apparent differences between the sub-groups containing patients with highly localized tumours, which were reported in our previous report, are now less marked and not statistically significant.