The apeE gene of Salmonella enterica serovar Typhimurium is induced by phosphate limitation and regulated by phoBR.

Mutations in apeR, a regulatory locus of the outer membrane esterase apeE from Salmonella enterica serovar Typhimurium, were shown to be alleles of the pstSCAB-phoU high-affinity phosphate transport operon. Expression of apeE was induced by phosphate limitation, and this induction required the phoBR phosphate regulatory system.

opdA, a Salmonella enterica serovar Typhimurium gene encoding a protease, is part of an operon regulated by heat shock.

The opdA (prlC) gene of Salmonella enterica serovar Typhimurium and Escherichia coli encodes the metalloprotease oligopeptidase A (OpdA). We report that opdA is cotranscribed with a downstream open reading frame, yhiQ. Transcription of this operon is induced after a temperature shift (30 to 42 degrees C), and this induction depends on the heat shock sigma factor encoded by the rpoH (htpR) gene.

The apeE gene of Salmonella typhimurium encodes an outer membrane esterase not present in Escherichia coli.

Salmonella typhimurium apeR mutations lead to overproduction of an outer membrane-associated N-acetyl phenylalanine beta-naphthyl ester-cleaving esterase that is encoded by the apeE gene (P. Collin-Osdoby and C. G. Miller, Mol. Gen. Genet. 243:674-680, 1994). This paper reports the cloning and nucleotide sequencing of the S. typhimurium apeE gene as well as some properties of the esterase that it encodes. The predicted product of apeE is a 69.9-kDa protein which is processed to a 67-kDa species by removal of a signal peptide. The predicted amino acid sequence of ApeE indicates that it is a member of the GDSL family of serine esterases/lipases. It is most similar to a lipase excreted by the entomopathogenic bacterium Photorhabdus luminescens. The Salmonella esterase catalyzes the hydrolysis of a variety of fatty acid naphthyl esters and of C6 to C16 fatty acid p-nitrophenyl esters but will not hydrolyze peptide bonds. A rapid diagnostic test reported to be useful in distinguishing Salmonella spp. from related organisms makes use of the ability of Salmonella to hydrolyze the chromogenic ester substrate methyl umbelliferyl caprylate. We report that the apeE gene product is the enzyme in Salmonella uniquely responsible for the hydrolysis of this substrate. Southern blot analysis indicates that Escherichia coli K-12 does not contain a close analog of apeE, and it appears that the apeE gene is contained in a region of DNA present in Salmonella but not in E. coli.

Epstein-Barr virus PCR correlated with viral histology and serology in pediatric liver transplant patients.

Epstein-Barr virus (EBV)-associated illnesses in posttransplant patients are difficult to diagnose. Attempts to aid in the diagnosis of such illnesses using the polymerase chain reaction (PCR) analysis for EBV have met with variable success due to the potential exquisite sensitivity of the assay. We have designed a relatively insensitive EBV PCR assay and compared the results with objective evidence of EBV activity including serologic response and in situ hybridization for the EBV genome. Eighty-five specimens from 65 patients were analyzed by the EBV PCR using DNA from whole blood. EBV serologic evaluation was done on 53 of the samples and in situ hybridization for EBV (EBER-1 mRNA) on 46 paired liver biopsies. Of 85 samples, 25 (29%) were positive for EBV using the PCR assay. Intensity of amplification was graded 0.5-1+ (weak) to 3+ (strong). Using these criteria, 19 EBV PCR-positive samples were graded 0.5-1+, 5 were graded 2+, and 1 was graded 3+. Of the moderate to strongly positive samples (2+ or 3+), five of six had two or more EBER-1-positive cells in the liver biopsies. Of the remaining 40 liver biopsies with either negative or weak positive PCR results, 3 had only single cells positive for EBER-1; the remainder were negative. In addition, PCR-positive results correlated with increasing EBV anti-early antigen antibody (P = .005) and viral capsid antigen IgG immunoglobulin G VCA (P = .05) EBV-positive results using the PCR assay correlated with objective evidence for increased EBV burden in children after liver transplantation. These preliminary data suggest that this PCR test may be useful to help guide immunosuppressive therapy in the posttransplant patient. Further evaluation using larger numbers of patients will be necessary to confirm this.

A comparison of wear patterns in vivo and in vitro for Björk-Shiley Delrin heart valve discs.

MATERIALS AND METHODS: Wear patterns for 33 explanted Björk-Shiley Delrin heart valve discs with known implant durations were compared and contrasted with the wear patterns for 17 discs from accelerated wear tested (AWT) valves. Previous test reports and the current study provided data for the maximum depth of wear grooves for a total of 56 explanted BSD valve discs with implant durations up to 22.4 years. RESULTS: The discs of the explanted BSD valves showed, in general, more than one type of wear pattern. Eighteen out of 33 exhibited a full indent groove on the inflow side and 24 of the 33 discs showed well indents on the outflow side. Only two discs showed evidence of non-concentric edge wear. All maintained their original surface polish outside of these areas. The data indicated that wear patterns in the case of the AWT discs were more pronounced, and in some discs were different in type from those of discs from explanted valves. For example, no cracks, fractures or perforations of the Delrin discs were observed, or reported, for the explanted valves, while in the 17 AWT valves there were three cracked discs, four disc fractures, three disc well perforations and three perforating edge indents. CONCLUSION: It appears that the Shiley AWT conditions were more severe and imposed greater loads than those produced by patients with BSD valves implanted.

Evaluation of disc retention of Björk-Shiley Delrin heart valves.

BACKGROUND AND AIMS OF THE STUDY: Impact wear grooves have been observed in the occluder discs of clinically explanted Björk-Shiley Delrin (BSD) heart valves due to repetitive closing impacts against the inlet strut. We examined whether this impact wear could increase the disc-to-strut gap, which could in turn affect the retention of occluder disc. MATERIALS AND METHODS: Three BSD heart valves with deep wear grooves were tested in a left ventricular assist device set-up simulating dynamic conditions that would facilitate the dislodgement. RESULTS: The disc was found to be rotating within the flange during the testing and no embolization of the occluder disc occurred in any of the BSD valves tested. The Björk retention limit and the physical retention limit were calculated for each valve. The Björk retention limit is defined as (disc well depth)-(disc-to strut gap)-(1/2 outlet strut wire diameter) and the physical retention as (disc well depth)-(disc-to strut gap). CONCLUSION: The disc retention would not be affected by the impact wear grooves as long as the disc-to-strut gap did not exceed the physical retention limit.

10 years of pediatric liver transplantation.

PURPOSE: Pediatric liver transplantation is an accepted therapy for end-stage liver disease, but little long-term data exist. METHODS: From October 1984 to October 1994, 202 patients underwent a total of 225 liver transplantations. There were 98 boys and 104 girls, the average age was 5.1 +/- 4.9 (range, 0.2 to 19.1) years. Thirty (16%) were under 1 year of age. The diseases that required transplantation included biliary atresia (BA) (45%), metabolic liver disease (MLD) (9.9%), acute hepatic failure (6.9%), and Alagille's syndrome (AS) (5.4%). Originally the immunosuppression was cyclosporine- and steroid-based; the later regimens also included azathioprine and antilymphocyte preparations. All reported survival rates were derived from life-table analysis. RESULTS: The patient survival rates at 1, 5, and 10 years were 76%, 70%, and 61%; the retransplantation rate was 11%. The respective graft survival rates were 71%, 63%, and 59%. There were 60 deaths; 48 (81%) occurred in the first year. These first-year deaths were from sepsis (20; 42%), central nervous system problems (5; 11%), intraoperative complications (4; 8%), lymphoproliferative disease (LPD) (2; 4%), rejection (2; 4%), primary nonfunction (2; 4%), and miscellaneous other causes (7; 15%). There were 12 deaths after the first year, from LPD (3; 25%), sepsis (1; 8%), rejection (2; 18%), cancer (1; 9%), secondary hepatic failure (1; 9%), cerebral vascular accident (1; 9%), or pre- or postoperative complications (3; 25%). Compared with the overall survival rate, patients with MLD had a better chance of survival (83%; P <.012) than did those with AS (45%; P < .001). The 5- and 10-year survival rates for patients with BA were 61% and 58%. Over the past 2 years, the survival rate has increased (87% v 72%; P < .05) as early septic deaths have decreased (from 2.6 to 1.0 per year). CONCLUSION: Liver transplantation is effective treatment for end-stage liver disease. Decreasing the number of early septic deaths has improved the chance of survival, and better diagnosis and treatment of LPD would improve the late survival rate.

A working cardiac valve phantom for radiographic assessment of prosthetic heart valves.

RATIONALE AND OBJECTIVES: A working valve phantom (WVP) that both exercises the valve occluder and simulates movements of the mitral annulus is described. It was designed to develop a method for radiographic detection of a single broken leg of the two-legged Björk-Shiley convexo-concave (C/C) heart valve outlet strut. METHODS: The WVP consists of a pneumatically driven left ventricular assist device immersed in 22 cm of water. Left ventricular assist device annulus movements are generated by systolic turgor and diastolic relaxation of the aortic outflow graft within limits set by the holding fixture design. RESULTS: WVP images were comparable in attenuation, valve motion, and diagnostic sensitivity to clinical C/C valve images and were effective in assessing leaflet excursions in another valve model. Techniques developed in the WVP have proved successful in the clinical detection of C/C valves that have a single broken leg but that show normal function in all other tests. CONCLUSION: The WVP can be a useful tool for developing refined radiographic assessments of prosthetic heart valves.

Extracorporeal liver assist device: hope for the future.

The liver has the unique property of regeneration in an acute setting if given enough time. Currently the only successful intervention for liver failure, both acute and chronic, is orthotopic liver transplantation. The increasing cost of transplantation and subsequent medical care, coupled with the decreasing availability of transplantable organs, magnifies the need for an alternative solution. A liver assist device that is metabolically similar to a native liver will advance the treatment regimen and decrease the need for surgery.

The Hepatix extracorporeal liver assist device: initial clinical experience.

Eleven patients were treated with the Hepatix extracorporeal liver assist device (ELAD) between June 1991 and August 1993. The first 2 patients were treated according to Food and Drug Administration guidelines ("Emergency Use of Unapproved Medical Devices," October 22, 1985), and the remaining 9 were treated according to an Investigational Device Exemption (IDE). The goal of the study was to establish the short-term safety of ELAD therapy, with a focus on acute medical complications such as hemodynamic instability, complement activation, and deterioration of vital organ function. As secondary goals, the metabolic capacity of ELAD cartridges and their clinical impact were assessed. Treatment was considered successful if the patient recovered sufficient liver function to survive weaning from the ELAD or was stabilized until orthotopic liver transplantation was performed. No short-term safety problems were associated with ELAD use. In addition, metabolic support was documented in 10 of the 11 patients, and 6 patients reached a successful end-point. The Hepatix ELAD is safe, and it provides measurable metabolic support in patients with late-stage liver failure. This pilot study provides the impetus to perform controlled trials of ELAD therapy in the treatment of various types of end-stage liver disease.

Cloning and nucleotide sequence of the cyclic AMP receptor protein-regulated Salmonella typhimurium pepE gene and crystallization of its product, an alpha-aspartyl dipeptidase.

The Salmonella typhimurium pepE gene, encoding an N-terminal-Asp-specific dipeptidase, has been cloned on pBR328 by complementation of the Asp-Pro growth defect conferred by a pepE mutation. Strains carrying the complementing plasmids greatly overproduce peptidase E. The enzyme has been purified from an extract of such a strain, its N-terminal amino acid sequence has been determined, and crystals suitable for X-ray diffraction have been grown. A new assay using L-aspartic acid p-nitroanilide as a substrate has been used to determine the pH optimum (approximately 7.5) and to test the effect of potential inhibitors. Insertions of transposon gamma delta (Tn1000) into one of the plasmids have been used to localize the gene and as sites for priming sequencing reactions. The nucleotide sequence of a 1,088-bp region of one of these plasmids has been determined. This sequence contains an open reading frame that predicts a 24.8-kDa protein with an N-terminal sequence that agrees with that determined for peptidase E. The predicted peptidase E amino acid sequence is not similar to that of any other known protein. The nucleotide sequence of the region upstream from pepE contains a promoter with a cyclic AMP receptor protein (CRP) site, and the effects of growth medium and of a crp mutation on expression of a pepE-lacZ fusion indicate that pepE is a member of the CRP regulon. The unique specificity of peptidase E and its lack of sequence similarity to any other peptidase suggest that this enzyme may be the prototype of a new class of peptidases. Its regulation by CPR and its specificity suggest that the enzyme may play a role in allowing the cell to use peptide aspartate to spare carbon otherwise required for the synthesis of the aspartate family of amino acids.

University of Wisconsin preservation solution compared with Euro Collins preservation solution in pediatric liver transplantation.

Orthotopic liver transplantation has become the accepted treatment for end-stage liver disease in children. To evaluate the efficacy of the University of Wisconsin (UW) preservation solution in pediatric liver transplantation, a group of 34 livers preserved with UW solution was compared in a nonrandomized fashion with a historical control group of 34 livers preserved with Euro Collins (EC) solution. Primary graft nonfunction did not occur in either group. Both groups were similar with respect to age, sex, weight, diagnosis, severity of the recipient's condition, donor condition at harvest, donor/recipient blood type match, and immunosuppressive management. The UW group had a significantly higher bilirubin, AST, ALT, and GGT during the first week after transplantation when compared with the EC group but no significant differences were noted after the ninth post-transplant day. No differences were noted when the groups were compared as to surgery time (9.1 v 8.4 hours), blood volumes replaced (1.8 v 2.0), number of ICU days (5.0 v 6.5), total number of infections per graft (1.0 v 0.8), total hospital days (31 v 30), and hospital cost ($134,000 v $126,000). The total preservation time was improved from 7 hours (range, 3.2 to 9.9) in the EC group to 13.9 hours (range, 6.9 to 22.3) in the UW group (P < .001). UW solution allows a significant increase in cold ischemic time in liver transplantation when compared with EC. This increase in preservation time resulted in no detrimental effect when compared with EC and potentially led to milder episodes of rejection in the postoperative period.