The solution structure of bacteriophage lambda protein W, a small morphogenetic protein possessing a novel fold.

Protein W (gpW) from bacteriophage lambda is required for the stabilization of DNA within the phage head and for attachment of tails onto the head during morphogenesis. Although comprised of only 68 residues, it likely interacts with at least two other proteins in the mature phage and with DNA. Thus, gpW is an intriguing subject for detailed structural studies. We have determined its solution structure using NMR spectroscopy and have found it to possesses a novel fold consisting of two alpha-helices and a single two-stranded beta-sheet arranged around a well-packed hydrophobic core. The 14 C-terminal residues of gpW, which are essential for function, are unstructured in solution.

Structural proteomics of an archaeon.

A set of 424 nonmembrane proteins from Methanobacterium thermoautotrophicum were cloned, expressed and purified for structural studies. Of these, approximately 20% were found to be suitable candidates for X-ray crystallographic or NMR spectroscopic analysis without further optimization of conditions, providing an estimate of the number of the most accessible structural targets in the proteome. A retrospective analysis of the experimental behavior of these proteins suggested some simple relations between sequence and solubility, implying that data bases of protein properties will be useful in optimizing high throughput strategies. Of the first 10 structures determined, several provided clues to biochemical functions that were not detectable from sequence analysis, and in many cases these putative functions could be readily confirmed by biochemical methods. This demonstrates that structural proteomics is feasible and can play a central role in functional genomics.

Thermodynamic and functional characterization of protein W from bacteriophage lambda. The three C-terminal residues are critical for activity.

Gene product W (gpW), the head-tail joining protein from bacteriophage lambda, provides a fascinating model for studying protein interactions. Composed of only 68 residues, it must interact with at least two other proteins in the phage, and probably with DNA. To study the structural and functional properties of gpW, plasmids were constructed expressing gpW with hexahistidine tag sequences at either the N or C terminus. The purified wild type fusion proteins were found to be stably folded and biologically active. The protein is monomeric as judged by equilibrium ultracentrifugation, and appears to unfold by a cooperative two-state mechanism. Circular dichroism studies indicate that the protein is 47% helical, with a T(m) of 71.3 degrees C, and a DeltaG(u) of 3.01 kcal/mol at 25 degrees C. Mutagenesis of the three hydrophobic C-terminal residues of gpW showed that they are critical for activity, even though they do not contribute to the thermodynamic stability of the protein. Using secondary structure prediction as a guide, we also designed destabilized gpW mutants. The hydrophobic nature of the gpW C terminus caused these mutants to be degraded by the ClpP-containing proteases in Escherichia coli.

A simple in vivo assay for increased protein solubility.

Low solubility is a major stumbling block in the detailed structural and functional characterization of many proteins and isolated protein domains. The production of some proteins in a soluble form may only be possible through alteration of their sequences by mutagenesis. The feasibility of this approach has been demonstrated in a number of cases where amino acid substitutions were shown to increase protein solubility without altering structure or function. However, identifying residues to mutagenize to increase solubility is difficult, especially in the absence of structural knowledge. For this reason, we have developed a method by which soluble mutants of an insoluble protein can be easily distinguished in vivo in Escherichia coli. This method is based on our observation that cells expressing fusions of an insoluble protein to chloramphenicol acetyltransferase (CAT) exhibit decreased resistance to chloramphenicol compared to fusions with soluble proteins. We found that a soluble mutant of an insoluble protein fused to CAT could be selected by plating on high levels of chloramphenicol.

Laparoscopic replacement of urinary tract segments using biodegradable materials in a large-animal model.

OBJECTIVE: We elected to evaluate laparoscopic segmental bladder and ureteral replacement with free biodegradable graft materials in a large-animal model. MATERIALS AND METHODS: In 18 Yucatan minipigs, a 1.5- to 2.8-cm segment of the upper ureter was excised. In 15 study animals, the ureter was laparoscopically replaced: by a stinted (6F double-J stent) tube graft made of acellular matrix (AMX) prepared from minipig ureters (MUMX) in 6 animals, acellular matrix prepared from domestic pig ureters (DUMX) in 3, and small-intestinal submucosa (SIS) in 6. In 3 control animals, the ureteral gap was bridged only by an indwelling stent. The stent was removed at 6 weeks, and retrograde ureteropyelography was performed preoperatively and at 8 and 12 weeks postoperatively, when animals were sacrificed. In 18 Yucatan minipigs, 3 x 3-cm bladder dome segments were laparoscopically replaced: by acellular matrix prepared from minipig small bowel (MBMX) in 6 animals, and SIS in 6 animals. The bladder was closed primarily in 6 control animals. Bladder capacity was assessed preoperatively and at 6 and at 12 weeks, when the animals were sacrificed. RESULTS: The average operating time for ureteral replacement was 187 (range 105-360) minutes. At 12 weeks, all animals had complete obstruction at the level of the replacement, with fibrosis +/- bone formation at the level of the stricture. For the bladder replacement groups, the average operating time was 147 (range 85-200) minutes. At 12 weeks, the bladder capacity was 60% of the preoperative capacity in the control group, 118% in the MBMX group, and 142% in the SIS group. Histologic examination showed regeneration of urothelium and some muscle with both MBMX and SIS. CONCLUSIONS: We were able to develop a reliable laparoscopic technique for both segmental ureteral and partial bladder replacement in a porcine model. With AMX and SIS replacement, regeneration of urothelium occurred in both ureter and bladder. However, functional replacement was successful only in the bladder.

Mechanism of ureteral stent flow: a comparative in vivo study.

BACKGROUND AND OBJECTIVES: The flow characteristics of ureteral stents have yet to be clearly defined. In this study, flow mechanics were studied in several silicone-based stents including 4.8F, 7F, and 10.3F pigtail; 7F Tower; and a prototype mesh stent. MATERIALS AND METHODS: Forty-five female Yucatan minipigs underwent bilateral laparoscopic occlusion of their renal vessels to stop urine production. A nephrostomy tract was established by retrograde puncture. A stent was placed in the ureter, and three measurements were taken with flow from a bag of irrigant 20 cm above the kidney: stent occluded with a guidewire (extraluminal flow), stent unobstructed (total flow), and laparoscopically placed extraureteral ligature (luminal flow). RESULTS: Luminal flow and, to a lesser extent, total flow appeared to increase as the internal and external diameters of the stent increased. The Tower stent, which had no sideholes, had much lower flow rates in all categories, while the prototype mesh stent showed greater total flow compared with the other stents. Extraluminal flow did not increase with stent size greater than 7F. CONCLUSIONS: Luminal flow, but not extraluminal flow, increased with an increase in the internal diameter of the stent. In general, the least favorable flow occurred with a Tower stent, which had the smallest internal diameter. The greatest flow was seen with the prototype mesh stent.

Mutagenesis of a buried polar interaction in an SH3 domain: sequence conservation provides the best prediction of stability effects.

The SH3 domain from the Fyn tyrosine kinase possesses a buried hydrogen bond between the side chains of a glutamate (Glu24) and a serine (Ser41) residue. Multiple amino acid substitutions were made at these positions to determine the role of this interaction in the stability and conformational specificity of the domain and to assess the relationship between the thermodynamic stability of mutants and sequence conservation seen in the SH3 domain family. Analysis of single and double alanine mutations indicated that the Glu24-Ser41 interaction contributes 0.50 kcal/mol to the stability of the domain. However, disruption of the Glu24-Ser41 interaction did not impair peptide binding function, suggesting that the interaction is not critical for conformational specificity. The stability of the domain was not increased by the replacement of these residues with different combinations of hydrophobic residues or with potential salt bridge forming residues. Despite their similar structural roles in the Fyn SH3 domain, the Ser41 position was considerably more tolerant to substitution than was the Glu24 position. An alignment of >350 different SH3 domains has been completed in our laboratory. A statistically significant correlation was found between the conservation data for the Glu24 and Ser41 positions and the thermodynamic stabilities of the mutants constructed at these positions. Surprisingly, our analysis of sequence alignment data provided a more accurate prediction of the stability of mutants than did examination of the three-dimensional structure of the domain.

Adult endopyelotomy: impact of etiology and antegrade versus retrograde approach on outcome.

PURPOSE: We evaluate our experience with endopyelotomy for ureteropelvic junction obstruction by stratifying the results of an antegrade versus a retrograde approach for primary, secondary, calculi related, high insertion and impaired renal function related obstruction, individually. MATERIALS AND METHODS: We retrospectively reviewed results of 149 nonrandomized patients treated for ureteropelvic junction obstruction, of whom 83 underwent antegrade percutaneous endopyelotomy using a right angle Greenwald electrode and 66 underwent retrograde endopyelotomy using a cutting balloon device. Subjective results were based on an analog pain scale, objective results on renal scan, excretory urography or Whitaker test and cost-effectiveness analysis on total treatment cost. RESULTS: In both primary and secondary ureteropelvic junction obstruction, retrograde endopyelotomy was related to a significantly shorter operating room time and hospital stay (p < 0.05). When treating noncalculous primary ureteropelvic junction obstruction (92 patients) there was a better objective, albeit not statistically significant, success rate with antegrade endopyelotomy (89 versus 71%) but retrograde endopyelotomy was 20% more cost-effective. When treating secondary ureteropelvic junction obstruction (37 patients) there was a better objective, albeit not statistically significant, success rate (83 versus 77%) with retrograde endopyelotomy, which was 37% more cost-effective. Complication rates were higher with antegrade compared to retrograde endopyelotomy for primary and secondary ureteropelvic junction obstruction (25 versus 14% and 26 versus 0%). In 20 patients with concomitant stones endopyelotomy results were better (93 to 100% success) than for any other categories of ureteropelvic junction obstruction. Of note, endopyelotomy also provided a reasonable outcome among patients with a high insertion primary ureteropelvic junction obstruction (70% success). CONCLUSIONS: Antegrade endopyelotomy is the preferred approach in patients with primary ureteropelvic junction obstruction and concomitant renal calculi (13.4% of cases), and may also be preferable in patients with high insertion obstruction (6.7%). For all other primary and all secondary ureteropelvic junction obstruction, antegrade and retrograde endopyelotomy is effective therapy yet retrograde endopyelotomy results in less operating room time, shorter hospital stay, fewer complications and significantly less expense to achieve the desired outcome.

Laparoscopic ureteroureterostomy using vascular closure staples in porcine model.

The greatest difficulty in performing a laparoscopic pyeloplasty is the suturing of the ureteropelvic junction. The purpose of this study was to evaluate the use of nonperforating titanium vascular closure staple (VCS) clips to perform in laparoscopic ureteroureterostomy in the porcine model. Six female minipigs underwent laparoscopic transection of one of the proximal ureters at the level of the lower pole of the kidney. Ureteroureterostomy was then performed using the titanium VCS clips. The animals were evaluated at 6 and 12 weeks postureteroureterostomy with retrograde pyelography and differential creatinine clearances. At 12 weeks, the animals were euthanized, and the area of ureteroureterostomy was examined grossly and histopathologically. The technique for laparoscopic vascular clipping of the ureteroureterostomy proved to be fast and effective. Follow-up indicated that the method was successful in producing a functionally patent anastomosis. No encrustation, stone formation, or intraluminal clip was noted in any of the ureters or kidneys undergoing the ureteroureterostomy. The area of the ureteroureterostomy showed minimal fibrosis and inflammation on histopathologic examination. In this animal study, the nonperforating titanium clips facilitated the performance of a laparoscopic ureteroureterostomy.

Endopyelotomy for high-insertion ureteropelvic junction obstruction.

We assessed the results of endourologic treatment of patients with a primary ureteropelvic junction obstruction (UPJO) caused by high insertion of the ureter into the renal pelvis (HIUPJO). A total of 10 patients 15 to 76 years old with preoperatively diagnosed HIUPJO were treated. Acucise retrograde endopyelotomy was performed in eight patients and percutaneous antegrade endopyelotomy in two. A stent was left in place for an average of 5.3 weeks. The subjective success rate, based on patient questionnaire and analog pain scales, was 80% at 27 months' average follow-up. The objective success rate, based on diuretic renal scanning or Whitaker test, was 70% at 26 months' mean follow-up. Overall, 60% of the patients had both an objectively and a subjectively successful outcome. The success rate for endopyelotomy in patients with UPJO caused by high insertion is similar to that reported for endopyelotomy in patients without high insertion. High insertion is not a contraindication to endopyelotomy.

Effect of mineral oil on porcine urothelium.

Mineral oil has been used to facilitate ureteral stone extraction and to treat selected patients with infected residual urine. The purpose of this study was to evaluate the effect of mineral oil on the urothelium. Twelve adult female farm pigs underwent bilateral ureteral catheter placement under general endotracheal anesthesia. Retrograde pyelograms were performed and the ureteral diameters measured. Using a randomization protocol, six animals underwent injection of 10 mL of normal saline into one ureteral catheter and 50 mL of normal saline instillation into the bladder. In the remaining six animals, 10 mL of mineral oil was injected into one ureteral catheter and 50 mL of mineral oil into the bladder. The instillation was maintained for 30 minutes, and then the catheters were removed. One week later, under general endotracheal anesthesia, cystoscopy and retrograde pyelography were performed to measure the diameter of the ureters, and the animals were euthanized. The bladder, ureters, and kidneys were harvested for macroscopic and histopathologic evaluation. There was no significant difference in the diameter of the ureters injected with normal saline, the uninjected ureters, or the mineral oil-injected ureters. The bladders, ureters, and kidneys were grossly normal in all animals. No significant histopathologic changes were noted in the ureteral or bladder urothelium or the renal parenchyma in the animals injected with mineral oil. In conclusion, the instillation of mineral oil within the urinary tract does not have any significant long-term functional or histopathologic effect on the urothelium.

Student evaluation in obstetrics and gynecology: self- versus departmental assessment.

Self-assessment of clerkship performance in obstetrics and gynecology was completed by 142 medical students and compared with departmental evaluations. Significant positive correlations existed between self- and departmental assessments for each of seven parameters in three categories of evaluation (clinical activities, written examination, and oral examinations). Students graded themselves lower than the actual faculty ratings and higher than the actual resident ratings. There was no difference in self- or departmental evaluations in regard to student gender. The amount of previous clerkship experience did not correlate with either the actual or perceived grades. Overall, self-assessments are consistent with those rendered by the department.