Characteristics and clinical significance of bacterial colonization of ureteral double-J stents in children.

OBJECTIVE: To determine the frequency, type of pathogen and clinical significance of bacterial colonization of double-J stents after pyeloplasty in children. PATIENTS AND METHODS: The medical files of 82 consecutive children (22 girls, 60 boys) who underwent pyeloplasty at a tertiary pediatric medical center in 2000-2007 were reviewed. Additional inclusion criteria were sterile urine preoperatively and placement of an indwelling double-J stent during surgery. Intravenous gentamicin was administered prior to pyeloplasty and stent removal; cephalexin was administered postoperatively until discharge. Children with a postoperative urinary tract infection (UTI) received full-dose antimicrobial treatment followed by prophylaxis until stent removal. RESULTS: Median patient age at surgery was 11 months (1 month-17.5 years). Forty-nine stents were inserted on the left side, 32 on the right, and one bilaterally. Cultures showed bacterial colonization in 58 cases (70.7%); 15 (25.8%) grew Staphylococcus (coagulase negative and positive). Eight children had febrile UTI postoperatively; in four the stent was colonized by Enterococci and in one by Proteus; three were sterile. There was no statistically significant association between positive stent culture and febrile UTI, patient age or sex, or stent laterality. The study was potentially limited by its observational design, small sample size, and the selective antibiotic treatment of patients with UTI which may have affected stent bacterial resistance. CONCLUSION: Bacterial colonization is not uncommon in double-J stents retained for several weeks after pyeloplasty, but is usually not clinically significant. Enterococcus is the most frequent pathogen.

Reimplantation of obstructive megaureters with and without tailoring.

OBJECTIVES: Reimplantation of megaureters may be complicated by excessive bulk of tissue when plicated, or stenosis at the anastomosis site when excisional tapering is performed. Some urologists in our institution reimplant megaureters without tailoring, while others routinely tailor megaureters. Aiming to compare success rates, we report our experience in reimplanting obstructive megaureters with and without tailoring. MATERIALS: The medical records of 30 boys and four girls with primary obstructive megaureters (a total of 38 ureters) were reviewed. Median age at operation was 18 months (range 1 month to 8 years). Of the ureters, 16/38 were tailored and 22/38 were reimplanted without tailoring. RESULTS: Preoperative mean diameter (per ultrasound) of the 16 tailored ureters was 16.1mm (range 10-26 mm). Postoperative ultrasound showed resolution of hydronephrosis in 3/16 (19%), an improvement in 11/16 (69%), and no change in the degree of hydronephrosis in 2/16 (13%). Preoperative mean diameter of the non-tailored ureters was 14 mm (range 10-27 mm), and postoperative ultrasound showed resolution of hydronephrosis in 11/22 (50%), an improvement in 10/22 (45%), and no change in the degree of hydronephrosis in 1/22 (5%). Of the tailored ureters, 1/16 needed re-operation because of anastomotic stricture, and one girl had recurrent urinary tract infections. Of the non-tailored ureters, four had recurrent infection, of whom one boy underwent redo reimplantation because of grade 2 vesicoureteric reflux. CONCLUSION: In this series non-tailoring reimplantation was an equivalent option to tailoring in obstructive megaureters.

Urethral polyps in prepubertal girls.

PURPOSE: Urethral polyps in girls are included in the differential diagnosis of interlabial masses. Only 6 cases have been described in the English literature to date and etiology is uncertain. We present 5 more cases and review the literature. We also propose an etiology for urethral polyps in young girls and compare polyps between girls and boys as well as older females. MATERIALS AND METHODS: The medical records of prepubertal girls with urethral polyps seen at our hospital between 1980 and 2003, including operative reports and followup, were reviewed. A senior pathologist re-inspected the specimens to confirm the diagnosis. A MEDLINE search of urethral polyps was performed. RESULTS: We identified 5 girls 2 to 10 years old with urethral polyps. All girls presented with "vaginal" bleeding, 1 had recurrent urinary tract infections and vulvitis, 1 had urinary frequency, and 1 had painful voiding. The polyps were not site specific and were located in the distal, mid and proximal urethra. All polyps were excised and were found benign. Four girls were cured after 1 procedure, while in one girl the polyp reappeared. The patients were followed for 1 to 17 years and eventually became asymptomatic. CONCLUSIONS: Urethral polyps in prepubertal girls are probably benign. We suggest that the etiology of most of polyps in girls is similar to those in older females, that is a prolapsing urothelium that has evolved into a polyp.

Management of post-operative bladder spasm.

OBJECTIVE: Pain management following bladder surgery in children is often complicated by bladder spasm. The overall severity of spasm can be reduced with opioids, anticholinergic medication and sedatives, although breakthrough spasms often occur. At the Royal Children's Hospital, Melbourne, intravesical bupivacaine has been used to manage postoperative bladder spasm to good effect. The administration of intravesical bupivacaine is analysed in this prospective audit of locally applied intravesical anaesthetic and compared with other methods. METHOD: From February to August 2003, histories of 58 patients who had intravesical bupivacaine were studied and compared with six other methods of management of postoperative bladder spasm. CONCLUSION: Data showed that epidural anaesthesia was the most effective treatment of pain, with a pain score reduction of 6.6, compared with a reduction of 6.1 with intravesical bupivacaine, and 4.5 using intravenous morphine. However, intravesical bupivacaine was the most effective method for the relief of bladder spasm.

Does the endoscopic technique of ureterocele incision matter?

PURPOSE: Endoscopic ureterocele decompression is a well established procedure in children. However, an accurate endoscopic incision may be challenging in large ectopic ureteroceles. We describe a percutaneously assisted technique to facilitate the ease of ureterocele incision and review other described methods. MATERIALS AND METHODS: We reviewed the medical records of 12 children with ectopic ureteroceles subtending a double collecting system who underwent endoscopic, percutaneously assisted incision. Six ureteroceles were on the left side, 5 were on the right side and 1 child had bilateral ureteroceles. Decompression results were evaluated by ultrasound and Tc-mercaptoacetyltriglycine imaging during a mean of 2.8 years of followup. RESULTS: There were 7 girls and 5 boys. Mean age at presentation was 11.6 months (range 1 week to 6 years). The decompression success rate was 84% (11 of 13 renal units), and improved renal function and drainage was noted in 5 of 12 patients (41.6%). Seven of 12 patients had vesicoureteral reflux, of whom 2 were asymptomatic at followup and, hence, were treated conservatively. Five children underwent surgery because of recurrent urinary tract infections. CONCLUSIONS: Although our results are similar to those of other methods, percutaneously assisted cystoscopic incision of ureterocele enables easier and more accurate decompression. However, when comparing the various techniques described, it seems that postoperative results mostly reflect the anatomical and functional characteristics of the urinary system rather than the technique used.

Uniquely conserved non-translated regions are involved in generation of the two major transcripts of protein phosphatase 2Cbeta.

Partial cDNAs of different isoforms of protein phosphatase 2Cbeta (PP2Cbeta or PPM1B) have been characterized in mammals. We disclose here the full cDNAs of two major PP2Cbeta isoforms from human, rat and mouse. These cDNAs (2.6 and 3.3 kb) are able to encode 53 kDa (PP2Cbetal) and 43 kDa (PP2Cbetas) polypeptides, respectively. The isoforms are co-expressed ubiquitously with the highest level in skeletal muscle, as assessed by Northern-blot analysis. Western and in situ analyses using monoclonal antibodies against PP2Cbeta confirmed the existence of two isoforms in the cytoplasm. Comparative sequence analysis revealed that both cDNAs consist of six exons with an alternate usage of the 3' exons that underlies the differences between them. The genomic structure of PP2Cbeta is similar to that of other PP2C paralogs and includes a non-coding first exon followed by a large intron and a large second exon that encoded most of the catalytic domain. Both variants of the ending exon include large non-coding regions. All non-translated regions (NTRs) are highly conserved between the orthologous genes, indicating their regulatory function. The 5'-NTR is long (379 bp), includes upstream start codons and is predicted to contain stable secondary structures. Such features inhibit translation initiation by the scanning mechanism. Introduction of this NTR element into a bi-luciferase expression-cassette enabled expression of the second cistron, suggesting that it might serve as an internal ribosome entry site, or it contains a cryptic promoter. Overexpression of PP2Cbeta under CMV-promoter in 293 cells led to cell-growth arrest or cell death.

Protein phosphatase 2Calpha expression in normal human tissues: an immunohistochemical study.

Protein phosphatase (PP2Calpha) is a member of the mammalian serine threonine-specific protein phosphatases family. We produced monoclonal antibodies against the recombinant PP2Calpha and evaluated the immunoreactivity of normal human tissues. The reactivity was strong in normal skin, the digestive tract, lung, kidney, breast, prostate, endocrine glands, and brain, while it was moderate in the ovary, testis, and liver. Epithelial cells revealed high levels of PP2Calpha expression, but stromal cells, including fibroblasts and endothelial cells, showed no or little PP2Calpha expression. Given the broad reactivity in endocrine and secreting epithelial cells, we propose that PP2Calpha expression might contribute to secretory cell function.

Inhibition of Streptomyces griseus aminopeptidase and effects of calcium ions on catalysis and binding--comparisons with the homologous enzyme Aeromonas proteolytica aminopeptidase.

Streptomyces griseus aminopeptidase is a zinc metalloenzyme containing 2 mol zinc/mol protein, similar to the homologous enzyme Aeromonas proteolytica aminopeptidase. In addition, a unique Ca2+-binding site has been identified in the Streptomyces enzyme, which is absent in the Aeromonas enzyme. Binding of Ca2+ enhances stability of the Streptomyces enzyme and modulates its activity and affinity towards substrates and inhibitors in a structure-dependent manner. Among the three hydrophobic 4-nitroanilides of alanine, valine and leucine, the latter displays the largest overall activation (increase in k(cat)/Km). Large enhancements in affinity (1/Ki) upon Ca2+ binding have been observed for inhibitors with flexible (leucine-like) residues at their N-termini and smaller enhancements for inhibitors with rigid (phenylalanine-like) residues.

Sensitive substrates for neprilysin (neutral endopeptidase) and thermolysin that are highly resistant to serine proteases.

Tripeptide derivatives like 3-carboxypropanoylalanyl-alanyl-leucine 4-nitroanilide or 3-carboxypropanoyl-alanyl-alanyl-phenylalanine 4-nitroanilide are very sensitive substrates for neprilysin (k cat > 10(2)s(-1); k cat/Km > or = 10(6) s(-1) x M(-1)) and are widely employed in investigations of the enzyme. However, these compounds are also good substrates for the serine proteases chymotrypsin and subtilisin (k cat approximately 1s(-1)-34s(-1)). By substituting the N-terminal alanine of the substrates with proline, the catalytic efficiency of the enzymic reaction, by the serine proteases, is diminished by 2-3 orders of magnitude, whereas that by neprilysin and theromlysin decreases only slightly. These effects demonstrate that structural alterations in peptide substrates that impair secondary sub-site interactions with one class of peptidases may enhance the selectivity of the substrates towards another class of peptidases.

Massive splenomegaly responsive to proguanil and with features of hairy cell leukaemia.

A recent Ethiopian immigrant to Israel presented with pneumococcal sepsis, massive splenomegaly and lymph-adenopathy. Investigations revealed many features of both hairy cell leukaemia (HCL) and hyperreactive malarious splenomegaly (HMS). Proguanil therapy for HMS was followed by rapid, marked decrease in spleen size, disappearance of the tartrate-resistant acid phosphatase-positive cells characteristic of HCL, and increasing eosinophilia, but unchanged lymphadenopathy.

Crystallization and preliminary crystallographic analysis of Streptomyces griseus aminopeptidase.

Streptomyces griseus excretes a small molecular mass (30 kDa) aminopeptidase that could be used for various biotechnological applications. This enzyme was isolated from an extracellular protease mixture of Streptomyces griseus (Pronase E. Sigma) and single crystals were obtained by the vapor diffusion method using polyethylene glycol 4000 as the precipitant. The crystals belong to the tetragonal space group P4(1)2(1)2 (P4(3)2(1)2), with cell dimensions of a = b = 61.82(3) A and c = 145.88(4) A. These crystals are mechanically strong, they are stable in the X-ray beam and they diffract to better than 1.8 A resolution. The cell dimensions and the cell symmetry are consistent with one molecule in the asymmetric unit and the crystals are suitable for a detailed high-resolution crystallographic analysis. A complete native data set to 1.9 A resolution has been collected on a Rigaku R-AXIS-IIC Imaging Plate Detector system and a heavy-atom derivative search is in progress.

Specificity of Streptomyces griseus aminopeptidase and modulation of activity by divalent metal ion binding and substitution.

Streptomyces griseus aminopeptidase is a calcium-activated zinc metalloenzyme characterized by a high enzymic reactivity, high thermal stability and low molecular mass [Spungin, A. and Blumberg, S. (1989) Eur. J. Biochem. 183, 471-477]. A study of the specificity of S. griseus aminopeptidase using amino acid 4-nitroanilide substrates shows that the leucine derivative is the best substrate. Derivatives of other hydrophobic amino acids, methionine and phenylalanine, are also excellent substrates for the enzyme. The 4-nitroanilides of alanine, valine, proline and lysine are good substrates whereas those of the small size glycine and the acidic amino acids are very poor. No hydrolysis of a terminal Xaa residue can be detected with Xaa-proline N-terminal sequences. Calcium ions bind to the enzyme and modulate its activity in a substrate-dependent manner. The catalytically essential zinc of S. griseus aminopeptidase is removed by dialysis against 1,10-phenanthroline and replaced by manganese or cobalt ions, resulting in enzyme derivatives of altered specificities. Thus, whereas the zinc enzyme hydrolyzes leucine 4-nitroanilide at a 10-fold faster rate than the manganese or cobalt enzymes, the cobalt enzyme hydrolyzes alanine 4-nitroanilide at a more than 20-fold faster rate than the zinc or manganese enzymes.

Investigation of neutral endopeptidases (EC 3.4.24.11) and of neutral proteinases (EC 3.4.24.4) using a new sensitive two-stage enzymatic reaction.

A sensitive two-stage enzymatic reaction for mammalian and bacterial metalloendopeptidases has been developed using the substrate 3-carboxypropanoyl-alanyl-alanyl-leucine-4-nitroanilide supplemented with Streptomyces griseus amino-peptidase. Neutral endopeptidase EC 3.4.24.11 from bovine kidney hydrolyzes the substrate (pH 7.5, 25 degrees C) with a catalytic efficiency (kcat = 1.2 x 10(2) s-1, Km = 0.15 mM) of the highest ever reported for the enzyme acting on synthetic chromophoric and fluorogenic substrates. Thermolysin hydrolyzes the substrate at a faster rate (kcat = 1.2 x 10(3) s-1) but the overall efficiency is diminished by a higher Km (4.2 mM). Suspensions of human neutrophil cells and culture filtrates of Bacillus cereus have been assayed sensitively for their neutral endopeptidases and neutral proteinase activities, respectively. The assay provides a convenient tool for the kinetic investigation of neutral endopeptidases and neutral proteinases and for assessing their function in biological systems.