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A patient developed severe ocular vaccinia via autoinoculation after acquiring unrecognized contact-transmitted vaccinia from wrestling with vaccinated members of his unit. This case highlights both the need to reinforce infection-control measures among vaccinees and the need for providers to be familiar with the identification and treatment of cutaneous and ocular vaccinia infection. [ABSTRACT FROM AUTHOR]

Purpose: To examine the long-term efficacy and safety of myopic implantable collamer lens (ICL) implantation in active duty personnel of U.S. military., Setting: Hospital practice., Design: Retrospective longitudinal observational study., Methods: 1485 patients (median age 25, interquartile range 22 to 29) underwent ICL surgery. Patients received a preoperative examination including uncorrected distance visual acuity (UDVA), intraocular pressure (IOP), manifest refraction measuring corrected distance visual acuity (CDVA), corneal topography and tomography, qualitative grading of perceived ectatic risk, ophthalmic biometry, and baseline endothelial cell counts (ECCs). Outcome measures included UDVA, IOP, vault size, manifest refraction, CDVA, and ECCs. The long-term follow-up data ware drawn from the U.S. military medical record system., Results: A total of 3105 eyes were evaluated. Patients received ICLs because of either abnormal topography (2111 eyes [68%]) or high myopia (994 eyes [32%]). 94 eyes (80%) maintained UDVA of 20/25 or better up to 8 years postoperatively. The rate of achieving the desired refractive correction was 97% (503 eyes) at 1 year and 90% (81 eyes) at 8 years. Stability of these outcomes was also shown by minimal change in manifest refraction. Documented mean ECC loss was 22% at postoperative year 5. The overall rate of adverse events was 1.2% (36 eyes) including visually significant cataract formation, glaucoma, retinal detachment, and traumatic incision opening. A removal or replacement rate of 4.5% (135 eyes) was observed., Conclusions: ICL implantation was found to be effective and safe. Vault sizes decreased over time, suggesting an increased risk of cataract formation after 7 years. Further study is necessary to assess long-term clinical significance of ECC decline., (Copyright © 2021 Published by Wolters Kluwer on behalf of ASCRS and ESCRS.)

Objectives: Postoperative bleeding is a common cause of morbidity and mortality in cardiac patients who undergo cardiopulmonary bypass (CPB). Pediatric patients are especially at risk for adverse effects of surgery and CPB on the coagulation system. This can result in bleeding, transfusions, and poor outcomes. Excessive bleeding unresponsive to blood products can warrant the off-label use of recombinant activated clotting factor VIIa (rFVIIa) and/or anti-inhibitor coagulant complex (FEIBA). Several studies have shown the utility in these agents off-label in patients who have undergone cardiac bypass surgery with acute bleeding episodes that are refractory to blood products. However, data regarding use of these agents in pediatrics are sparse. The purpose of this study is to report the use of rFVIIa and FEIBA in pediatric cardiac surgery patients in our institution., Methods: This was a retrospective chart review of pediatric cardiothoracic surgery patients who received rFVIIa or FEIBA at Children's Healthcare of Atlanta during the study period., Results: Thirty-three patients received rFVIIa and 9 patients received FEIBA either intraoperatively or postoperatively for bleeding related to the cardiac procedure. Approximately 13% of rFVIIa patients and 55% of FEIBA patients required repeat doses. There were decreases for all blood products administered after rFVIIa and FEIBA were given. However, the doses used did not correlate with either positive or negative outcomes. Seventeen percent (n = 7) of rFVIIa patients experienced a thrombus and 22% (n = 2) of FEIBA patients experienced a thrombus., Conclusions: Both rFVIIa and FEIBA reduced blood product usage in pediatric patients following cardiac procedures., Competing Interests: Disclosures The authors declare no conflicts or financial interest in any product or service mentioned in the manuscript, including grants, equipment, medications, employment, gifts, and honoraria. The authors had full access to all data and take responsibility for the integrity and accuracy of the data analysis., (Copyright Pediatric Pharmacy Association. All rights reserved. For permissions, email: mhelms@pediatricpharmacy.org 2020.)

Effective diabetes problem solving requires identification of risk factors for inadequate mealtime self-management. Ecological momentary assessment was used to enhance identification of factors hypothesized to impact self-management. Adolescents with type 1 diabetes participated in a feasibility trial for a mobile app called MyDay. Meals, mealtime insulin, self-monitored blood glucose, and psychosocial and contextual data were obtained for 30 days. Using 1472 assessments, mixed-effects between-subjects analyses showed that social context, location, and mealtime were associated with missed self-monitored blood glucose. Stress, energy, mood, and fatigue were associated with missed insulin. Within-subjects analyses indicated that all factors were associated with both self-management tasks. Intraclass correlations showed within-subjects accounted for the majority of variance. The ecological momentary assessment method provided specific targets for improving self-management problem solving, phenotyping, or integration within just-in-time adaptive interventions., (© The Author(s) 2019. Published by Oxford University Press on behalf of the American Medical Informatics Association. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

A major driver of wildlife responses to climate change will include non-genomic effects, like those mediated through parental behavior and physiology (i.e., parental effects). Parental effects can influence lifetime reproductive success and survival, and thus population-level processes. However, the extent to which parental effects will contribute to population persistence or declines in response to climate change is not well understood. These effects may be substantial for species that exhibit extensive parental care behaviors, like birds. Environmental temperature is important in shaping avian incubation behavior, and these factors interact to determine the thermal conditions embryos are exposed to during development, and subsequently avian phenotypes and secondary sex ratios. In this article, we argue that incubation behavior may be an important mediator of avian responses to climate change, we compare incubation strategies of two species adapted to different thermal environments nesting in extreme heat, and we present a simple model that estimates changes in egg temperature based on these incubation patterns and predicted increases in maximum daily air temperature. We demonstrate that the predicted increase in air temperature by 2100 in the central USA will increase temperatures that eggs experience during afternoon off-bouts and the proportion of nests exposed to lethal temperatures. To better understand how species and local adaptations and behavioral-plasticity of incubation behavior will contribute to population responses to climate change comparisons are needed across more avian populations, species, and thermal landscapes., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology. All rights reserved. For permissions please email: journals.permissions@oup.com.)

Purpose: To review data on malpractice claims related to refractive surgery to identify common allegations and injuries and financial outcomes., Methods: The WestlawNext database was reviewed for all malpractice lawsuits/settlements related to refractive eye surgery. Data evaluated included patient demographics, type of operation performed, plaintiff allegation, nature of injury, and litigation outcomes., Results: A total of 167 cases met the inclusion criteria, of which 108 cases (64.7%) were found to be favorable and 59 cases (35.3%) unfavorable to the defendant. A total of 141 cases were tried by a jury with 108 cases (76.4%) favorable and 33 cases (23.6%) unfavorable to the defendant. Laser in situ keratomileusis was performed in 127 cases (76%). The most common allegations were negligence in treatment or surgery in 127 cases (76%) and lack of informed consent in 83 cases (49.7%). For all cases, the need for future surgery (P = 0.0001) and surgery resulting in keratoconus (P = 0.05) were more likely to favor the plaintiff. In jury verdict decisions, cases in which failure to diagnose a preoperative condition was alleged favored the defendant (P = 0.03), whereas machine malfunction (P = 0.05) favored the plaintiff. After adjustment for inflation, the overall mean award was $1,287,872. Jury verdicts and settlements led to mean awards of $1,604,801 and $826,883, respectively., Conclusions: Malpractice litigation in refractive surgery tends to favor the defendant. However, large awards and settlements were given in cases that were favorable to the plaintiff. The need for future surgery and surgery leading to keratoconus increased the chance of an unfavorable outcome.

Before 1995, race D of Phytophthora phaseoli, the causal agent of downy mildew on lima bean (Phaseolus lunatus), was the prevalent physiological race in the mid-Atlantic region of the United States. Since 1995, however, new physiological races of P. phaseoli have been responsible for downy mildew outbreaks in previously resistant cultivars in this region. Cultivar differential testing of 180 isolates of P. phaseoli collected between 1994 and 2005 from Delaware and the eastern shore of Maryland has confirmed the presence of two new physiological races. The detection of race E in 1995 and race F only 5 years later in 2000, plus the lack of resistant cultivars to manage the epiphytotics in lima bean, have led to millions of dollars of crop losses. Intra- and interspecific genetic variation of Phytophthora spp. and isolates were assessed using amplified fragment length polymorphism DNA fingerprinting. Primer groups EcoRI+AG and MseI+C distinguished P. phaseoli and P. capsici from P. infestans but did not distinguish among different races of P. phaseoli.

Phytophthora capsici Leonian, the causal agent of lima bean pod rot, was first identified as a pathogen of lima bean in 2002 (1) and poses a new threat to lima bean (Phaseolus lunatus L.) production in the Mid-Atlantic Region. The phenylamide fungicide mefenoxam (Ridomil Gold; Syngenta Crop Protection) is widely used in the region for controlling foliar and soilborne diseases caused by Oomycetes. Isolates of P. capsici were collected from lima bean pods from production fields in Delaware, Maryland, and New Jersey from 1998 to 2004. These isolates originated from survey samples of lima bean fields for another pathogen, P. phaseoli, in 1999 and 2000 and diagnostic samples were submitted to the Plant Disease Clinic. Isolates were from lima bean, except for one from pepper (basal stem). Identification was made on the basis of morphometric characteristics. No known sensitive or insensitive isolates were included in the evaluation. Single zoospore cultures were evaluated for mefenoxam sensitivity on V8 agar plates amended with 100 ppm of mefenoxam, a previously tested concentration (2). Seven-millimeter-diameter agar plugs of each isolate were cut from the edge of actively expanding cultures of P. capsici with a cork borer and transferred to three V8 agar plates amended with mefenoxam and three unamended V8 plates. The plates were arranged in a completely randomized design and incubated at 25°C in the dark for 3 days. After incubation, colony growth was measured in millimeters and averaged for the three replicate plates of each isolate and percent growth relative to the unamended control was calculated. Mefenoxam sensitivity was assigned according to methods of Lamour et al. (2). The experiment was repeated once, and also run with a treatment of 200 ppm of mefenoxam. Of sixteen isolates screened, nine were rated as sensitive, four were intermediately resistant, and three were resistant. There was no difference between the 100 and 200 ppm results, except for a slight increase in sensitivity for one isolate. A subsequent experiment tested five isolates at concentrations of 1, 10, 100, and 1,000 ppm. Results were consistent with previous tests, with resistant isolates exhibiting some growth at the highest concentration of mefenoxam. One resistant isolate was from a field in Delaware previously cropped to slicing cucumbers with a history of mefenoxam applications. The second was from Caroline County, MD, which is heavily cropped to pickling cucumbers and likely to have been exposed to mefanoxam applications for the control of fruit rot; the origin of the third insensitive isolate from lima bean is unknown. Mefanoxam usage on lima bean is usually limited to one foliar application of mefenoxam+copper hydroxide to control downy mildew in the fall crop in wet seasons. This study indicates that mefenoxam resistance is present in populations of P. capsici in lima bean fields in the Mid-Atlantic Region, presumably as a result of mefenoxam applications to other vegetable crops, principally cucurbits, which are planted in rotation with lima beans or from nearby cucurbit fields. Implementing strategies to minimize fungicide resistance in other vegetables is important to slow resistance development associated with this emerging pathogen on lima beans. Lima bean pod rot continues to be seen sporadically each year in fields with a history of P. capsici and abundant rainfall or excessive irrigation. References: (1) C. R. Davidson et al. Plant Dis. 86:1049, 2002. (2) K. H. Lamour et al. Phytopathology 90:396, 2000.

Purpose: To document the incidence and treatment of patients with severe ocular and ocular adnexal injuries during Operation Iraqi Freedom., Design: Retrospective hospital-based observational analysis of injuries., Participants: All coalition forces, enemy prisoners of war, and civilians with severe ocular and ocular adnexal injuries., Methods: The authors retrospectively examined severe ocular and ocular adnexal injuries that were treated by United States Army ophthalmologists during the war in Iraq from March 2003 through December 2005., Main Outcome Measures: Incidence, causes, and treatment of severe ocular and ocular adnexal injuries., Results: During the time data were gathered, 797 severe eye injuries were treated. The most common cause of the eye injuries was explosions with fragmentation injury. Among those injured, there were 438 open globe injuries, of which 49 were bilateral. A total of 116 eyes were removed (enucleation, evisceration, or exenteration), of which 6 patients required bilateral enucleation. Injuries to other body systems were common., Conclusions: Severe eye injuries represent a significant form of trauma encountered in Operation Iraqi Freedom. These injuries were most commonly caused by explosion trauma.

We have shown previously that cytoplasmic p53 is covalently linked to 5.8S rRNA. The covalent complex is associated with a small subset of polyribosomes, which includes polyribosomes translating p53 mRNA. Because 5.8S rRNA resides in or near the ribosomal P site, our findings suggested involvement of p53 in translational regulation. Ninety-seven kiloDaltons eEF2 was found to coimmunoprecipitate in a salt-stable complex with p53. The 97 kDa species was identified as eEF2, because it was (1) recognized by a polyclonal antiserum specific for eEF2, (2) ADP-ribosylated by diphtheria toxin (DT), and (3) radiolabeled by gamma-32P-azido-GTP and UV-irradiation. p53 and eEF2 sedimented in sucrose gradients in both polyribosomal and subribosomal fractions. Subribosomal p53 can bind eEF2 without the mediation of ribosomes, because (1) it binds subribososomal eEF2, (2) it binds phosphorylated eEF2, and (3) subribosomal p53-bound eEF2 can be ADP-ribosylated by DT. No effect of p53 activation was found on eEF2 expression or phosphorylation. However, the binding of eEF2 to p53 decreased when cytoplasmic p53 migrated to the nucleus. Renaturation of temperature sensitive A135V mutant p53 (ts-p53) was found to alter the sensitivity of p53 mRNA translation, but not bulk mRNA translation, to the translocation-specific elongation inhibitor, cycloheximide (Cx). The association of p53 with two translational components involved in ribosomal translocation, eEF2 and 5.8S rRNA, and the effect of p53 on sensitivity to the translocation inhibitor, Cx, as well as the known molecular interactions of these components in the ribosome suggest involvement of p53 in elongation., (Copyright 2003 Wiley-Liss, Inc.)

Lima beans are an important crop in Delaware and the Mid-Atlantic Region. In the summer of 2000, five commercial cultivars (3-28, 184-85, C-elite Sel, Butter Bean, and Jackson Wonder) of lima bean in Delaware, Maryland, and New Jersey were observed with white, appressed mycelia on infected pods that appeared distinctly different from signs of downy mildew infection caused by Phytophthora phaseoli. Isolations were made by placing diseased pods between layers of rye media (1). A fungus that produced white mycelia with sporangia was consistently isolated. All Phytophthora isolates from the infected pods were heterothallic, grew at 35°C, had as much as 100 μm long pedicles on varying shapes of caducous sporangia with tapering base and >2 papillae, and were identified as P. capsici (2). Initially, three surface-disinfected pods from cv. Early Thorogreen plants grown in the greenhouse were floated on 20 ml of sterile water in a petri dish, and each was inoculated with a disk of P. capsici. This was repeated for nine isolates obtained from lima bean. After incubation for 7 days at room temperature, all 27 pods were infected, and P. capsici was reisolated from all the pods. A pathogenicity test was performed on the same cultivars from which the original field isolates were collected. Three seedlings and two plants with mature pods were inoculated with a sporangial suspension of each of the nine isolates and placed in a dew chamber for 5 days at 20 to 25°C and 100% relative humidity. White mycelial growth was observed on seedlings and mature pods. One inoculated plant developed brown-to-black stem lesions with white mycelia. All pods on the mature plants showed appressed, white mycelia identical to that observed in the commercial lima bean fields. P. capsici was consistently reisolated from all inoculated plants. In 2000, most infected pods in infested fields were observed low in the plant canopy or touching the soil. However, in 2001, infected pods were mostly in the lower and mid-portion of the plants observed in baby lima bean fields in Kent County, DE. References: (1) C. E. Caten and J. L. Jinks. Can. J. Bot. 46:329, 1967. (2) D. C. Erwin and O. K. Ribeiro. Phytophthora capsici. Page 264 in: Phytophthora Diseases Worldwide. The American Phytopathological Society, St Paul, MN, 1996.

Adequate anterior segment visualization during cataract surgery can be hindered when excessive tear film secretions precipitate on the corneal surface before the initial corneal incision is made. In most cases, room-temperature balanced salt solution applied to the corneal surface clears the debris. However, in cases in which tear film precipitates persist after the use of room-temperature balanced salt solution, the application of warm balanced salt solution can provide rapid and sustained dispersion of the precipitates. We present our experience using this technique.

Downy mildew, incited by Phytophthora phaseoli Thaxt., is the most important disease of lima bean (Phaseolus lunatus L.) on the east coast of the United States. It has been a serious threat to commercial lima bean production in Delaware, Maryland, and New Jersey for the past 5 years. Growers have attempted to manage this disease using resistant cultivars and copper hydroxide fungicides. In August and September 1995, a new pathogenic race of P. phaseoli was isolated from infected pods of the lima bean cv. Packer in a production field near Milton, DE. Races of P. phaseoli are determined using a modification of a cultivar differential developed by Wester (3). The cv. 184-85, which is resistant to races A, B, C, and D (1), is susceptible to the new race, designated as E. In August 2000, another new pathogenic race of P. phaseoli was isolated from infected pods of cv. 184-85 near Middletown, DE. The lima bean line BG2-408, which is resistant to races A, B, C, D, and E, is susceptible to the new race, designated as F. Symptoms produced on lima bean plants infected by races E and F are similar to each other, and to those produced by all other races. All races of P. phaseoli have the same cultural characteristics on lima bean pod agar. Evaluations of in field weather station data and disease occurrence indicate that races E and F may have temperature maxima greater than 32°C, whereas race D has a maximum of less than 32°C (2). During the 2000 growing season, 118 isolates of P. phaseoli were collected from 44 production fields in Delaware and the eastern shore of Maryland, with 86% characterized as race E and 5% as race F. References: (1) C. R. Davidson et al. Biol. Cult. Tests 2001:V80. (2) R. A. Hyre and R. S. Cox. Phytopathology 43:419, 1953. (3) R. E. Wester. Phytopathology 60:1856, 1970.

A cogent update of orbital blowout history, anatomy, and management are included with a retrospective study of 59 pure orbital blowout fractures which occurred between 1994 and 1998. Our goal is to provide a better understanding of this frequently encountered entity and to help augment the confidence of nonophthalmologists who will often evaluate patients with suspected orbital blowout fractures.

ABSRACT In 1996 to 1998, a late-blight survey was conducted in potato- and tomato-growing regions of Morocco. A total of 149 isolates of Phytophthora infestans were collected and analyzed for the glucose-6-phosphate isomerase (Gpi) and peptidase (Pep) alleles, mating types, and metalaxyl sensitivities. Four genotypes were identified: MO-1 (mating type A1, Gpi 100/100, Pep 92/100), MO-2 (mating type A1, Gpi 86/100, Pep 92/100), MO-3 (mating type A2 Gpi 100/100, Pep 100/100), and MO-4 (mating type A1, Gpi 100/100, Pep 100/100). The potato isolates were MO-1 (1996 & 97), MO-3 (1998), and MO-4 (1998). The frequencies of A1 (MO-4) and A2 (MO-3) mating types in potato fields in 1998 were 26 and 74%, respectively. Potato isolates were pathogenic to both potatoes and tomatoes. The isolates collected from tomatoes in 1997 and 1998 were MO-2. Potato and tomato isolates were insensitive and sensitive to metalaxyl, respectively. The change of genotype population in 1998 was probably caused by migration of a new genotype from Europe associated with importation of potato seed. The detection of A1 and A2 mating types in the same potato field indicates the potential for sexual reproduction of P. infestans in Morocco.

Pathogenic strains of Phytophthora infestans insensitive to phenylamide have been reported from around the world and are responsible for a lack of late blight control in both potatoes and tomatoes. In vivo laboratory assay methods used to determine the sensitivity of P. infestans to fungicides include floating of leaflet disks on fungicide suspension and the use of potato tuber disks. In our study, these two methods were compared with a new detached-leaf assay. The fungicides chlorothalonil (protective), oxadixyl and metalaxyl (systemic), and cymoxanil (locally systemic) were utilized. Phenylamide-insensitive and -sensitive genotypes of P. infestans were used as inoculum. Significant differences between preventive and systemic fungicides were found, and all assay methods differentiated between phenylamide-sensitive and -insensitive genotypes of P. infestans. Differences among assays were significant when comparing mean inhibitory values. There was no significant difference between the detached leaf and the tuber disk assay, but both methods were significantly different from the floating-leaflet disk assay.

Greenhouse and field tests were conducted in 1992 and 1993 to determine the causal pathogen(s) of red root rot (RRR) of corn. Corn hybrids Dekalb DK 522, DK 572, DK 677, and DK 582 were utilized. Phoma terrestris, Pythium irregulare, and Fusarium acuminatum were used alone or in combination to infest potting mix in greenhouse tests or soil in field tests. Results indicated that P. terrestris is the primary pathogen in the RRR complex of corn in Delaware. When P. terrestris and Pythium irregulare were associated, the disease progressed faster and was more severe, resulting in significantly higher (P ≤ 0.01) root rot, basal stalk rot, and wilt. F. acuminatum played a minor role in causing the disease, with little additional symptom development when combined with P. terrestris or Pythium irregulare.

Prior to 1995, late blight of potatoes (Solanum tuberosum L.) caused by Phytophthora infestans (Mont.) de Bary had not been reported from Delaware for decades. In that year, the fungus was isolated from potato foliage collected from fields in the Dover area that sustained severe foliage loss due to the disease. To establish pathogenicity, potato and tomato plants grown in the greenhouse were inoculated with the isolates obtained and Koch's postulates were completed. Analyses to determine mating type, genotype at two allozyme loci (2), Gpi and Pep, and sensitivity to metalaxyl were also completed. Measurements were made every 5 days and EC 50 values were calculated for radial mycelial growth on metalaxyl-amended agar, for leaflets inoculated with P. infestans floated in metalaxyl solutions, and for inoculated potato tuber disks deposited on filter paper saturated with metalaxyl (1). Isolates were pathogenic to potatoes and tomatoes, were A2 mating type with Gpi 100/111/122 and Pep 100/100 (same as US-8), and displayed resistance to metalaxyl up to 140 mg/l. References: (1) K. L. Deahl et al. Am. Potato J. 70:779, 1993. (2) S. B. Goodwin et al. Plant Dis. 79:1181, 1995.

Late blight of potato (Solanum tuberosum L.) caused by Phytophthora infestans (Mont.) de Bary first appeared in Africa in 1941. It has been observed sporadically in Morocco for decades but only recently became a major problem. Significant losses have been recorded in the last two growing seasons in spite of the use of various disease control programs that included combinations of systemic and protectant fungicides. Phytophthora infestans was cultured from diseased foliage collected from commercial potato fields near Larache, Morocco. Isolates were analyzed to determine pathogenicity on several potato and tomato cultivars, mating type, genotype at two allozyme loci (2), and relative sensitivity to metalaxyl. Responses of the isolates to metalaxyl were assayed by mycelial radial growth on metalaxyl-amended agar, by floating leaves inoculated with P. infestans on metalaxyl solutions, and via potato tuber disks placed on filter paper saturated with metalaxyl solutions (1). Koch's postulates were completed; all isolates were pathogenic to potato and tomato cultivars tested, are consistent with the A1 mating type, and have the same allozyme pattern (Gpi 100/100, Pep 92/100) as US-6 genotype. All tests indicated resistance to metalaxyl up to 250 ppm. References: (1) K. L. Deahl et al. Am. Potato J. 70:779, 1993. (2) S. B. Goodwin et al. Plant Dis. 79:1181, 1995.

Our previous finding that the tumor suppressor p53 is covalently linked to 5.8S rRNA suggested functional association of p53 polypeptide with ribosomes. p53 polypeptide is expressed at low basal levels in the cytoplasm of normal growing cells in the G1 phase of the cell cycle. We report here that cytoplasmic wild-type p53 polypeptide from both rat embryo fibroblasts and MCF7 cells and the A135V transforming mutant p53 polypeptide were found associated with ribosomes to various extents. Treatment of cytoplasmic extracts with RNase or puromycin in the presence of high salt, both of which are known to disrupt ribosomal function, dissociated p53 polypeptide from the ribosomes. In immunoprecipitates of p53 polypeptide-associated ribosomes, 5.8S rRNA was detectable only after proteinase K treatment, indicating all of the 5.8S rRNA in p53-associated ribosomes is covalently linked to protein. While 5.8S rRNA linked to protein was found in the immunoprecipitates of either wild-type or A135V mutant p53 polypeptide associated with ribosomes, little 5.8S rRNA was found in the immunoprecipitates of the slowly sedimenting p53 polypeptide, which was not associated with ribosomes. In contrast, 5.8S rRNA was liberated from bulk ribosomes by 1% sodium dodecyl sulfate, without digestion with proteinase K, indicating that these ribosomes contain 5.8S rRNA, which is not linked to protein. Immunoprecipitation of p53 polypeptide coprecipitated a small fraction of ribosomes. p53 mRNA immunoprecipitated with cytoplasmic p53 polypeptide, while GAPDH mRNA did not. These results show that cytoplasmic p53 polypeptide is associated with a subset of ribosomes, having covalently modified 5.8S rRNA.

Red root rot of corn (Zea mays L.) has caused sporadic disease losses in Delmarva during the past 6 years. A survey was made between 21 August and 14 September 1995 in 11 counties in Delaware and on the eastern shore of Maryland to detect Phoma terrestris (E. M. Hans.) in corn roots and in the surrounding soil. Sampling was based on one field per 1,200 ha of corn production in each county. Three root samples and one soil sample were collected from each of 100 corn production fields from corn plants with diseased basal stalk tissue. The presence of P. terrestris was confirmed by cultures from root samples that produced a beet-red color on Watson's sterile wheat straw agar (2). Serial dilutions of soil samples in 0.1% water agar were mixed with unsolidified Watson's basal medium, followed by amendment with a 2-(4-thiazolyl)benzimidazole solution (1). P. terrestris was detected in root samples from 99% of the fields surveyed and from 82% of the soil samples. Soil populations ranged from 0.2 to 200 CFU per g of soil, with a mean of 12.5 CFU. This survey indicated that P. terrestris is widespread in corn production fields in Delaware and on the eastern shore of Maryland. Disease symptoms in surveyed fields indicated that pathogenicity on corn is variable, generally does not result in serious losses, and is related to hybrid susceptibility and growing conditions. References: (1) B. Sneh et al. Phytopathology 64:275, 1974. (2) R. D. Watson. Plant Dis. Rep. 45:289, 1961.

Twelve patients with recurrent, metastatic, or inoperable gastric adenocarcinoma were enrolled in an Illinois Cancer Center phase II trial of amonafide (nafidimide), a novel compound that acts as a DNA intercalator. Treatment consisted of a 60-minute infusion of amonafide which was administered daily for 5 consecutive days every 3 weeks at a starting dose of 300 mg/m2/d. Doses were modified according to the grade of toxicity experienced and eight patients underwent dose escalations. All 12 patients were evaluable for response and toxicities were predominantly hematologic. Stabilization of disease for at least 28 days was observed in seven patients and disease progression was noted in five. The median survival was 7.4 months. Doses were sufficient to produce severe bone marrow toxicity in one-third of the patients treated. None of the patients responded to therapy, implying a true response rate less than .221. Based on the results of this study, amonafide showed no activity against gastric adenocarcinoma; however toxicity appeared acceptable at the 300 mg/m2/d x 5 consecutive days every 3 weeks dose and schedule.

We report here the isolation and identification of the RNA specifically immunoprecipitated and covalently linked to the tumor suppressor gene product p53. After treatment with proteinase K, the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) band of p53 yields a single, discrete 157-nucleotide RNA, which was cloned, sequenced, and identified as 5.8S rRNA. 5.8S rRNA was obtained only after proteolysis of the p53 SDS-PAGE band. Free 5.8S rRNA did not comigrate with p53 in SDS-PAGE. This RNA was only immunoprecipitated from cells containing p53. Protein-free RNA obtained by proteolysis of the p53 band hybridized to the single-stranded DNA vector containing the antisense sequence of 5.8S rRNA. The covalence of the p53-5.8S rRNA linkage was demonstrated by the following findings: (i) p53 and the linked 5.8S rRNA comigrated in SDS-PAGE; (ii) only after treatment of the p53-RNA complex with proteinase K did the 5.8S rRNA migrate differently from p53-linked 5.8S rRNA; and (iii) this isolated RNA was found linked to phosphoserine, presumably at the 5' end. Covalent linkage to the single, specific RNA suggests that p53 may be involved in regulating the expression or function of 5.8S rRNA.

We have previously shown that the carboxyl-terminal tryptic peptide of the tumor suppressor p53 coeluted from reverse-phase high-performance liquid chromatography (HPLC) with ribonucleotides, suggesting the possible linkage of RNA to p53. In this report, we establish that p53 is covalently linked to RNA, using biochemical criteria at the levels of both tryptic peptide and intact protein: the electrophoretic properties of a tryptic peptide containing phosphorylated Ser-389 and the HPLC chromatographic properties of p53 depend on the linked RNA, p53, purified through urea-sodium dodecyl sulfate-polyacrylamide gel electrophoresis and HPLC, copurifies with RNA, and Ser-389 liberates ribonucleotides upon RNase or alkali treatment. Wild-type and mutant p53s from both simian virus 40 (SV40)-transformed and SV40-nontransformed cells are RNA linked, indicating that RNA linkage may be a general property of p53. The RNA is labeled in vivo with 3H-uridine and in vitro by RNA ligase, suggesting that the RNA is bound by a 5' linkage. The RNA is a long-lived, integral component of p53 rather than a transient reaction intermediate. RNA linkage occurs at an evolutionarily conserved site on p53. We propose that RNA-linked p53 is a major biologically active form of p53 and that its interaction with RNA-linked SV40 T antigen reflects a role in RNA metabolism.

The T antigen of simian virus 40, which may play a role in the control of viral DNA replication, is recovered from nuclei of cells transformed by simian virus 40 in several forms sedimenting at different rates. The large molecular weight forms are converted to the smallest (5 S) form by high salt, suggesting that they differ in the degree of aggregation. All the forms of the antigen bind efficiently to double-stranded DNA-cellulose columns at pH 6.2 and low salt, and elute in two fractions: one at pH 8.0 and low salt, the other at pH 8.0 and high salt. The antigen has little affinity for single-stranded DNA.

Newly synthesized simian virus 40 large tumor antigen (T Ag) slowly forms a stable complex with the host tumor antigen, "p53." By the use of immunological and temporal separations and inhibition of aggregation and processing by A locus mutation, we have distinguished specific steps in the reaction sequence leading to formation of the rapidly sedimenting oligomeric complex. The monoclonal antibody PAb101 bound only a fraction of the total soluble pulse-labeled T Ag bound by antitumor serum. After a chase, all T Ag had matured to the form recognized by PAb101. All p53 in the mouse line SVA31E7 was precipitated by the T Ag-specific monoclonal antibody PAb101, even after a short pulse, and is therefore entirely bound to mature T Ag. The p53-specific monoclonal antibody PAb122 precipitates nearly all of the mature T Ag recognized by PAb101, except A locus mutant T Ag, synthesized at the nonpermissive temperature. A locus mutation inhibited entry of newly synthesized T Ag into the oligomeric greater than 28S complex of T Ag and p53.

T-antigens from simian virus 40 (SV 40)-transformed and lytically infected cells have been isolated by immunoprecipitation and their molecular weights estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. T-antigen from SV40-transformed mouse and hamster cells has an apparent molecular weight of 94,000 whereas that from several lines of SV40-infected monkey cells is 84,000. In a wheat germ cell-free system, mRNA from either transformed or productively infected cells is translated into a 94,000 species. Experiments with the protease inhibitors L-l-(tosylamide-2-phenyl)ethylchloromethyl ketone HCl and N-alpha-p-tosyl-L-lysylchloromethyl ketone HCl suggest that the 84,000 species of T-antigen found in infected cells is derived from the larger species by proteolytic cleavage. Further, the cleavage pathway probably involves a two-step reaction with an 89,000 intermediate. The biological significance of the two molecular weight forms of T-antigen is unknown, but the possibility that they have different physiological activities is discussed.

RNA is shown to be covalently linked to the large tumor antigen (TAg) of simian virus 40 (SV40). Proteolytic digestion of TAg, isolated in the presence of ribonuclease inhibitors from SV40 transformed Balb/c mouse cells, generated a specific phosphopeptide of high charge heterogeneity that was strongly retained on DEAE-cellulose in the presence of 7 M urea. Hydrolysis of this peptide with RNAase released the four standard ribonucleotide monophosphates. Analysis of peptide digestion products showed that the RNA is attached to TAg through a phosphodiester linkage between the beta-hydroxyl of a serine and the 5' phosphate of an invariant cytidine residue. The methods applied to SV40 TAg can be applied to other proteins, including cellular oncogene products, to investigate the possibility of covalent protein-RNA interactions.

Two new families of host proteins (Mr, 48,000 and 55,000), in additional to the viral large (T) and small tumor antigens, are precipitable, with anti-T antiserum, from cells transformed or infected by the DNA tumor virus simian virus 40 (SV40). Rabbit anti-mouse 48,000 protein antiserum reacts specifically with SV40-infected or -transformed mouse cells to give nuclear staining indistinguishable from T-antigen staining but does not react with SV40-transformed human cells which nevertheless have structurally analogous 48,000 proteins, nor does it give nuclear fluorescence with untransformed mouse cells. Comparison of the partial proteolytic digests of the 48,000 proteins from cultured cells of various mammalian species shows that they are structurally related but not related to the 55,000 or large T-antigen proteins. The 55,000 proteins from the various mammalian species were also structurally related.

The oncogene product p53, isolated from SV3T3 cells where it forms a complex with simian virus 40 large tumor antigen (T antigen) in the nucleus, has been found to be phosphorylated at at least four distinct sites on the 390 amino acid protein. Separation of tryptic phosphopeptides has permitted identification of two sites as Ser-312 and Ser-389, and permitted analysis of the types of phosphate bonds. The peptide containing Ser-312 separates electrophoretically into three charged forms; two are resistant to dephosphorylation by both alkaline phosphatase and alkaline hydrolysis, suggesting a phosphodiester. The carboxyl-terminal phosphopeptide containing Ser-389 was alkaline phosphatase-resistant and liberated four ribonucleoside monophosphates upon base or RNase hydrolysis, suggesting that Ser-389 may be covalently linked to RNA. Phosphorylation of Ser-389 decreased markedly at the nonpermissive temperature in simian virus 40 tsA58-transformed cells, indicating a dependence on native T antigen function and a possible role in transformation by T antigen. Two additional phosphorylation sites, one involving serine and one involving threonine, probably reside in the amino-terminal segment of p53 and appear to be peptide-phosphate monoesters.

Simian virus 40 large tumor antigen (T Ag) can be separated by sucrose gradient sedimentation into a rapidly sedimenting, maximally phosphorylated fraction and a slowly sedimenting, less phosphorylated fraction. The Mr 48,000 host tumor antigen (48,000 HTA, also called nonviral T Ag) is preferentially complexed with the maximally phosphorylated T Ag. Pulse-labeled T Ag sediments as a 5-6S monomer, whereas T Ag radiolabeled for progressively longer periods slowly increases in sedimentation coefficient to give a broad distribution between 5 S and greater than 28 S. Mutation in the viral A locus causes a decrease in T Ag phosphorylation and a marked decrease in 48,000 HTA binding, shifting the sedimentation coefficient of T Ag to the monomer value. The more highly phosphorylated T Ag also has the highest affinity for chromatin.

Seventy-five percent of the amino acid sequence of simian virus 40 (SV40) large T antigen is identical to that of the large T antigen of the human papovavirus, BK virus (BKV). Cross-reactivity between BKV T antigen and monoclonal antibodies directed against SV40 T antigen was studied by immunofluorescence, an enzyme immunoassay, immunoprecipitation of radiolabeled extracts, and Western blotting. BKV T antigen was found to be recognized by two monoclonal antibodies, PAb 416 and 430, which react with two distinct sites toward the amino terminus of SV40 large T antigen. These two sites may correspond to two hydrophilic regions of shared amino acid sequence which exist toward the amino termini of the T antigens.

The self-association of gene-32 protein has been studied by sedimentation equilibrium centrifugation and polyacrylamide gel electrophoresis, in order to better understand its role in DNA replication and genetic recombination. The monomer molecular weight of gene-32 protein is 38,000 in guanidine hydrochloride and 34,000 in sodium dodecyl sulfate, in agreement with the results of Alberts and coworkers. Stable dimers of gene-32 protein occur under various conditions, among which are high ionic strength and pH 10. The occurrence of stable dimers under some conditions and higher aggregates under others indicates there are two types of protein-protein interactions occurring in gene-32 protein self-association. The association that occurs above about 0.1 mg/ml concentration of protein produces at least decamers.A model for the DNA replication fork is postulated that requires the two different interactions that occur in gene-32 protein aggregation. In the model, gene-32 protein holds the two strands of the DNA duplex in a conformation that prevents their reannealing and, therefore, facilitates replication and recombination.