dCtBP-dependent and -independent repression activities of the Drosophila Knirps protein.

Transcriptional repressor proteins play essential roles in controlling the correct temporal and spatial patterns of gene expression in Drosophila melanogaster embryogenesis. Repressors such as Knirps, Krüppel, and Snail mediate short-range repression and interact with the dCtBP corepressor. The mechanism by which short-range repressors block transcription is not well understood; therefore, we have undertaken a detailed structure-function analysis of the Knirps protein. To provide a physiological setting for measurement of repression, the activities of endogenous or chimeric Knirps repressor proteins were assayed on integrated reporter genes in transgenic embryos. Two distinct repression functions were identified in Knirps. One repression activity depends on dCtBP binding, and this function maps to a C-terminal region of Knirps that contains a dCtBP binding motif. In addition, an N-terminal region was identified that represses in a CtBP mutant background and does not bind to the dCtBP protein in vitro. Although the dCtBP protein is important for Knirps activity on some genes, one endogenous target of the Knirps protein, the even-skipped stripe 3 enhancer, is not derepressed in a CtBP mutant. These results indicate that Knirps can utilize two different pathways to mediate transcriptional repression and suggest that the phenomenon of short-range repression may be a combination of independent activities.

The effect of endoscopic sphincterotomy on acute and chronic complications of biliary endoprostheses.

BACKGROUND: Endoscopically placed biliary stents have become routine therapy for bile duct obstruction and bile leaks. Controversy exists regarding the use of biliary sphincterotomy to facilitate placement of 10F plastic stents. METHODS: We retrospectively studied the effect of sphincterotomy on acute and chronic complications of 10F stent therapy. Data for acute complications, 30-day mortality and stent migration were obtained for 130 patients undergoing placement of a single 10F plastic biliary stent. For 109 patients in whom prolonged stent therapy was undertaken, the occurrence of and time to stent dysfunction were also analyzed. Sphincterotomy was performed in 48 cases (36.9%) based on physician preference. RESULTS: There were no failures in stent placement. The incidence of acute complications was higher in patients undergoing sphincterotomy (8.3% vs. 1.2%, p = 0.04). Stent migration was more common in the no sphincterotomy group versus the sphincterotomy group (8.5% vs. 0, p = 0.03). CONCLUSIONS: Sphincterotomy is not necessary for placement of 10F plastic stents and increases acute procedural morbidity. Interestingly, a higher incidence of stent migration was seen in patients who did not undergo biliary sphincterotomy.

Transcriptional repression by the Drosophila giant protein: cis element positioning provides an alternative means of interpreting an effector gradient.

Early developmental patterning of the Drosophila embryo is driven by the activities of a diverse set of maternally and zygotically derived transcription factors, including repressors encoded by gap genes such as Krüppel, knirps, giant and the mesoderm-specific snail. The mechanism of repression by gap transcription factors is not well understood at a molecular level. Initial characterization of these transcription factors suggests that they act as short-range repressors, interfering with the activity of enhancer or promoter elements 50 to 100 bp away. To better understand the molecular mechanism of short-range repression, we have investigated the properties of the Giant gap protein. We tested the ability of endogenous Giant to repress when bound close to the transcriptional initiation site and found that Giant effectively represses a heterologous promoter when binding sites are located at -55 bp with respect to the start of transcription. Consistent with its role as a short-range repressor, as the binding sites are moved to more distal locations, repression is diminished. Rather than exhibiting a sharp 'step-function' drop-off in activity, however, repression is progressively restricted to areas of highest Giant concentration. Less than a two-fold difference in Giant protein concentration is sufficient to determine a change in transcriptional status of a target gene. This effect demonstrates that Giant protein gradients can be differentially interpreted by target promoters, depending on the exact location of the Giant binding sites within the gene. Thus, in addition to binding site affinity and number, cis element positioning within a promoter can affect the response of a gene to a repressor gradient. We also demonstrate that a chimeric Gal4-Giant protein lacking the basic/zipper domain can specifically repress reporter genes, suggesting that the Giant effector domain is an autonomous repression domain.

The FIS protein fails to block the binding of DnaA protein to oriC, the Escherichia coli chromosomal origin.

The Escherichia coli chromosomal origin contains several bindings sites for factor for inversion stimulation (FIS), a protein originally identified to be required for DNA inversion by the Hin and Gin recombinases. The primary FIS binding site is close to two central DnaA boxes that are bound by DnaA protein to initiate chromosomal replication. Because of the close proximity of this FIS site to the two DnaA boxes, we performed in situ footprinting with 1, 10-phenanthroline-copper of complexes formed with FIS and DnaA protein that were separated by native gel electrophoresis. These studies show that the binding of FIS to the primary FIS site did not block the binding of DnaA protein to DnaA boxes R2 and R3. Also, FIS appeared to be bound more stably to oriC than DnaA protein, as deduced by its reduced rate of dissociation from a restriction fragment containing oriC . Under conditions in which FIS was stably bound to the primary FIS site, it did not inhibit oriC plasmid replication in reconstituted replication systems. Inhibition, observed only at high levels of FIS, was due to absorption by FIS binding of the negative superhelicity of the oriC plasmid that is essential for the initiation process.

The pathologic measurement of polyp size is preferable to the endoscopic estimate.

BACKGROUND: There is no standardized technique to measure polyp size. Estimation of polyp size at endoscopy is difficult. Polyp size measurement by pathologists would seem to be an accurate alternative, but tissue fixation may alter polyp size. To evaluate methods of determining polyp size, we compared endoscopists' estimates and pathologists' measurements with measurements made by an independent examiner. METHODS: Polyps were measured by an independent investigator before and after formalin fixation. The investigator's measurement before fixation (the "gold standard") was compared with the endoscopists' estimates and the pathologists' measurements. RESULTS: Ten endoscopists removed 61 polyps with a snare in 33 patients: 82% were adenomatous and 72% were pedunculated. Mean size was 0.85 +/- 0.6 cm (SD) (range: 0.3 to 3.6 cm, 26% > or = 1 cm). Polyps remained in formalin for a mean of 239 minutes (46 to 1164 minutes). Polyps neither consistently shrank nor enlarged in formalin (maximum change +/- 0.2 cm, r = 0.99 [p < 0.001]). Interobserver agreement between pathologists' and the investigator's post-formalin measurements showed that 55 of 57 polyps (97%) were within +/- 0.3 cm. Endoscopists inaccurately estimated 11 of 56 polyps (20%) (> 0.3 cm difference from the independent examiner). Polyp size was underestimated in three instances (range 0.5 to 0.9 cm) and overestimated in eight (range 0.4 to 0.8 cm). In 5 of 11 instances (46%), this inaccuracy altered polyp size classification across the 1 cm threshold. Results were not dependent on endoscopist, histology, or polyp location. CONCLUSIONS: (1) Polyp size is not significantly affected by formalin fixation; 2) Endoscopists' estimates of polyp size are often unreliable; and, when possible, (3) Pathologists' measurements of polyp size should be used in clinical trials and in clinical practice.

Domains of DnaA protein involved in interaction with DnaB protein, and in unwinding the Escherichia coli chromosomal origin.

DnaA protein of Escherichia coli is a sequence-specific DNA-binding protein required for the initiation of DNA replication from the chromosomal origin, oriC. It is also required for replication of several plasmids including pSC101, F, P-1, and R6K. A collection of monoclonal antibodies to DnaA protein has been produced and the primary epitopes recognized by them have been determined. These antibodies have also been examined for the ability to inhibit activities of DNA binding, ATP binding, unwinding of oriC, and replication of both an oriC plasmid, and an M13 single-stranded DNA with a proposed hairpin structure containing a DnaA protein-binding site. Replication of the latter DNA is dependent on DnaA protein by a mechanism termed ABC priming. These studies suggest regions of DnaA protein involved in interaction with DnaB protein, and in unwinding of oriC, or low-affinity binding of ATP.

Ordered and sequential binding of DnaA protein to oriC, the chromosomal origin of Escherichia coli.

DnaA protein of Escherichia coli acts in initiation of chromosomal DNA replication by binding specific sequences, termed DnaA boxes in the chromosomal origin, oriC. On binding, it induces a localized unwinding to create a structure recognized by other replication proteins that act subsequently in the initiation process. In this report, we examined the binding of DnaA protein to each of the DnaA boxes in oriC. By gel mobility shift assays, DnaA protein formed at least six discrete complexes. ATP or ADP included in the reaction mixture prior to electrophoresis was required. Chemical cleavage of isolated complexes with 1,10-phenanthroline-copper revealed that DnaA protein binds in an ordered manner to the DnaA boxes in oriC. Preferential binding to one DnaA box (R4) was confirmed by demonstration that a DNA fragment containing it was bound with greater affinity than another DnaA box sequence (R1). In vitro replication activity correlated with a complex formed at a ratio of 30 DnaA monomers/oriC in which all DnaA boxes are occupied. The last site bound is DnaA box R3. This event may be critical in promoting initiation from oriC as it correlates with in vivo observations that binding of DnaA protein to box R3 occurs at the time of initiation of chromosomal replication, whereas other DnaA boxes are bound by DnaA protein throughout the cell cycle (Cassler, M. R., Grimwade, J. E., and Leonard, A. C.(1995) EMBO J. 14, 5833-5841).

How accurate are endoscopic estimates of size?

The accuracy of polyp size estimations in clinical studies is not known. This study was designed to evaluate how well endoscopists can estimate the size of objects at endoscopy. Observations were made by six attending gastroenterologists, six gastroenterology fellows, and seven untrained medical residents. Ball bearings ranging in size from 3 mm to 19 mm were randomly inserted into a latex colon model, and size was estimated while being viewed with a video colonoscope with and without the aid of an open biopsy forceps. Estimated size correlated well to actual size (R ranged from 0.78 to 0.93), although the mean estimates were consistently lower (13% to 29%) than the actual size for all groups, with and without forceps. The use of forceps did not improve the estimates. Exactly correct estimates occurred in 8%; errors of as much as 110% were recorded. The 12 mm bearing was estimated to be less than 10 mm half of the time. We conclude that endoscopists frequently underestimate the size of objects viewed at the time of endoscopy. There was no difference in the performance of experienced gastroenterologists, fellows in training, or untrained residents. This study indicates that better methods of training and for determining size are needed. Research that depends on endoscopic estimates of polyp size may be biased.

High resolution analysis of ground foot reaction forces.

A new system of hardware and software has been designed for quantitative high resolution analysis of ground foot pressures during stance and gait. In the system, a large photoelastic sheet is used as the transducer and a computerized video system for analysis and display. This system offers a larger active surface at higher resolution and at lower cost than any other system heretofore described. It will be used to predict diabetic plantar ulceration as well as to detect anatomic and motor defects affecting the feet.

Interaction of hemolysis and genotype on ionized calcium in bile of mice with hemolysis-induced gallstones.

In this study, we used an ion-selective membrane electrode to measure ionized calcium in hepatic bile of control +/+ mice and nb/nb mice with hereditary hemolytic anemia. We found that biliary concentrations of ionized, bound, and total calcium were significantly higher (p less than 0.001) and magnesium was significantly lower (p less than 0.001) in nb/nb mice than in control +/+ mice. To separate the hemolytic process from genotypic influences, we transplanted genetically defective bone marrow from nb/nb mice into histocompatible nonhemolytic recipients (W/Wv). After successful engraftment, transplanted W/Wv mice had significantly higher biliary concentrations of ionized calcium than their untreated W/Wv counterparts (p less than 0.001); but bound and total calcium and magnesium concentrations were not different from untreated W/Wv controls. When compared with nb/nb mice, transplanted W/Wv mice had lower ionized calcium (p less than 0.001) and higher bound calcium concentrations (p less than 0.001) in their biles. These data indicate that ionized calcium in hepatic bile is significantly influenced by genotypic factors and subsequently increased in chronic hemolysis; and further, that increased ionized calcium in bile of mice with hemolysis is a risk factor, but of limited predictive value for hemolysis-induced gallstone formation.