Ectopic pelvic spleen.

We are reporting the first, to our knowledge, ectopic pelvic spleen demonstrated preoperatively by means of a liver-spleen scan and a selective splenic arteriogram. Symptoms, consisting of crampy pain in the lower part of the abdomen exacerbated by standing or stooping, were relieved by splenectomy in this 19-year-old woman. Splenopexy, which has been advocated in the past, has no place in the management of this rare and interesting congenital variant. Laxity or failure of development of the phrenicosplenic and gastrosplenic ligaments is thought to account for the splenic descensus.

Role of liver scanning in the diagnosis of hepatic metastases.

Liver scanning is quite useful in detecting metastases, particularly when none are suspected clinically, and obviates the need for more elaborate and expensive examinations in about half the cases in the series analyzed here.

Validating self reported home safety practices in a culturally diverse non-inner city population.

OBJECTIVE: To determine the validity of face to face, self reported responses to questions about the presence of safety devices and use of safety practices in the home aimed at preventing unintended injuries to preschool aged children. METHODS: The authors invited families with children enrolling in a medium sized Midwestern US community Head Start program to participate in a randomized study of home safety practices. Participation involved consenting parents (n = 452) completing an initial questionnaire during Head Start enrollment or in their home. Project staff conducted home inspections to confirm parental responses to 16 questions. Only inspections conducted within 34 days of questionnaire completion (n = 259) were included in the validation study. Parents were told that the home visit would assess the need for safety devices, but were not informed of the validation aspect of the study. RESULTS: Sensitivities were generally high for all 16 safety practices, whereas negative predictive value and specificity varied considerably. Positive predictive value was also high for most practices, and did not vary by ethnicity. Answers provided by parents in their home were different and more reliable than those provided by parents interviewed at school (p = 0.001). CONCLUSIONS: Use of safety devices and practices by parents of preschool aged children reported in a face to face interview are generally reliable. Reliability increases if the interview is conducted in the home. Parents may also be more willing to report potential problems if they perceive they may receive corrective assistance.

Use of the modified Delphi technique to identify and rate home injury hazard risks and prevention methods for young children.

OBJECTIVE: For children aged 1-5 years, the authors used the Delphi method to determine (1) the most important injury hazards in each area of the home; (2) the most important injury prevention behaviors; and (3) feasible and efficacious safety devices and behaviors to reduce injury risks. DESIGN: The authors used a modified Delphi method to prioritize home injury hazards for children 1-5 years of age. The Delphi method is an indirect, anonymous, iterative process aimed at achieving consensus among experts; in this study, the authors queried key informants electronically. Thirty four key informants, primarily from the United States, participated in at least one of the three rounds of questionnaires. Responses were submitted by email or fax. Participants identified, rated, and ranked home injury hazards and prevention methods. RESULTS: The overall response rate for each survey ranged from 82% to 97%. Initially, 330 unique hazards and prevention behaviors/devices were identified in seven areas of the home. The 126 home injury hazards were rated based on frequency, severity, and preventability of injury; and the 204 behaviors and devices were rated by efficacy and feasibility. These experts rated firearms and pools as the most significant hazards, and smoke alarms and safe water temperature as the most important preventions. CONCLUSIONS: The modified Delphi method of consensus was useful to prioritize home injury hazards and prevention methods for children under the age of 6 years.

Multiple forms of ubiquitin-activating enzyme E1 from wheat. Identification of an essential cysteine by in vitro mutagenesis.

Ubiquitin-activating enzyme, E1, directs the ATP-dependent formation of a thiol ester linkage between itself and ubiquitin. The energy in this bond is ultimately used to attach ubiquitin to various intracellular proteins. We previously reported the isolation of multiple E1s from wheat and the characterization of a cDNA encoding this protein (UBA1). We now report the derived amino acid sequence of two additional members of this gene family (UBA2 and UBA3). Whereas the amino acid sequence of UBA2 is nearly identical to UBA1, the sequence of UBA3 is significantly different. Nevertheless, the protein encoded by UBA3 catalyzes the ATP-dependent activation of ubiquitin in vitro. Comparison of derived amino acid sequences of genes encoding E1 from plant, yeast, and animal tissues revealed 5 conserved cysteine residues, with one potentially involved in thiol ester bond formation. To identify this essential residue, codons corresponding to each of the 5 cysteines in UBA1 were individually altered using site-directed mutagenesis. The mutagenized enzymes were expressed in Escherichia coli and assayed for their ability to activate ubiquitin. Only substitution of the cysteine at position 626 abolishes E1 activity, suggesting that this residue forms the thiol ester linkage with ubiquitin.

Cloning of ubiquitin activating enzyme from wheat and expression of a functional protein in Escherichia coli.

The initial step in the conjugation of ubiquitin to substrate proteins involves the activation of ubiquitin by ubiquitin activating enzyme, E1. Previously, we purified and characterized multiple species of E1 from wheat germ. We now describe the isolation and characterization of a cDNA clone encoding E1 from wheat. This clone (UBA1) was isolated from a cDNA expression library with anti-wheat E1 antibodies. It contained an open reading frame coding for 1051 amino acids and directed the synthesis of a protein that comigrated with a wheat germ E1 of 117 kDa. UBA1 was confirmed as encoding E1 by (i) comparison of the peptide map of the protein product of UBA1 synthesized in Escherichia coli with that of purified E1 from wheat, and (ii) amino acid sequence identity of peptides generated from purified E1 with regions of the derived amino acid sequence of UBA1. The isolation of two additional cDNAs closely related to UBA1 indicated that E1 was encoded by a small gene family in wheat. Nonetheless, a single poly(A+) mRNA size class of 4 kilobases hybridized with UBA1. When expressed in E. coli, the product of UBA1 catalyzed the formation of a thiol ester linkage between ubiquitin and an ubiquitin carrier protein. The ability of E. coli containing UBA1 to synthesize an active protein will allow us to identify domains important for E1 function using in vitro mutagenesis.

The ubiquitin-activating enzyme (E1) gene family in Arabidopsis thaliana.

Conjugation of multiple ubiquitins serves as a committed step in the degradation of a variety of intracellular eukaryotic proteins by the 26S proteasome. Conjugates are formed via a three-enzyme cascade; the initial step requires ubiquitin-activating enzyme (E1), which couples ubiquitin activation to ATP hydrolysis. Previously, we showed that many higher plants contain multiple E1 proteins and described several E1 genes from wheat. To facilitate understanding of the roles of the different plant E1s, we characterized the E1 gene and protein family from Arabidopsis thaliana. Arabidopsis E1s are encoded by two genes (AtUBA1 and AtUBA2) that synthesize approximately 123-kDa proteins with 81% amino acid sequence identity to each other and 44-75% sequence identity with confirmed E1s from other organisms. Like other E1 proteins, AtUBA1 and 2 contain a cysteine residue in the putative active site for forming the ubiquitin thiol-ester intermediate. Enzymatic analysis of the corresponding proteins expressed in Escherichia coli demonstrated that both proteins activate ubiquitin in an ATP-dependent reaction and transfer the activated ubiquitin to a variety of Arabidopsis E2s with near equal specificity. Expression studies by quantitative RT-PCR and histochemistry with transgenic plants containing AtUBA promoter-beta-glucuronidase-coding region fusions showed that the AtUBA1 and 2 genes are co-expressed in most, if not all, Arabidopsis tissues and cells. Collectively, the data indicate that E1 proteins, and presumably the rest of the ubiquitin pathway, are present throughout Arabidopsis. They also show that the AtUBA1 and 2 genes are not differentially expressed nor do they encode E1s with dramatically distinct enzymatic properties.

Phytochrome A overexpression in transgenic tobacco. Correlation of dwarf phenotype with high concentrations of phytochrome in vascular tissue and attenuated gibberellin levels.

Phytochromes are a family of related chromoproteins that regulate photomorphogenesis in plants. Ectopic overexpression of the phytochrome A in several plant species has pleiotropic effects, including substantial dwarfing, increased pigmentation, and delayed leaf senescence. We show here that the dwarf response is related to a reduction in active gibberellins (GAs) in tobacco (Nicotiana tabacum) overexpressing oat phytochrome A under the control of the cauliflower mosaic virus (CaMV) 35S promoter and can be suppressed by foliar applications of gibberellic acid. In transgenic seedlings, high concentrations of oat phytochrome A were detected in stem and petiole vascular tissue (consistent with the activity of the CaMV 35S promoter), implicating vascular tissue as a potential site of phytochrome A action. To examine the efficacy of this cellular site, oat phytochrome A was also expressed using Arabidopsis chlorophyll a/b-binding protein (CAB) and the Arabidopsis ubiquitin (UBQ1) promoters. Neither promoter was as effective as CaMV 35S in expressing phytochrome in vascular tissue or in inducing the dwarf phenotype. Collectively, these data indicate that the spatial distribution of ectopic phytochrome is important in eliciting the dwarf response and suggest that the phenotype is invoked by elevated levels of the far-red-absorbing form of phytochrome within vascular tissue repressing GA biosynthesis.

Comparison of the three-dimensional structures of human, yeast, and oat ubiquitin.

The crystal structure of human ubiquitin has been solved by x-ray diffraction methods and refined by standard procedures to a conventional crystallographic R factor of 0.176 at 1.8-A resolution (Vijay-Kumar, S., Bugg, C.E., and Cook, W.J. (1987) J. Mol. Biol. 194, 525-538). Crystals of yeast and oat ubiquitin have been grown using human ubiquitin crystals as seeds. Diffraction data for yeast and oat ubiquitin have been collected to a resolution of 1.9 and 1.8 A, respectively. Difference Fourier electron-density maps reveal that the structures of yeast and oat ubiquitin are quite similar to human ubiquitin. All the amino acid changes are clustered in two small patches on one surface of the molecule. This surface is probably not involved in conjugation with proteins destined for ATP-dependent proteolysis.