A comparison between vapor tunnel and virtual basket for the treatment of proximal ureteral stones using holmium:YAG laser (Cyber Ho): which is the best tool to reduce retropulsion?

PURPOSE: To compare vapor tunnel (VT) and virtual basket (VB) tools to reduce retropulsion in the treatment of proximal ureteral stones. METHODS: Patients with a single proximal ureteral stone were randomly assigned to holmium laser lithotripsy with the use of VT (Group A) or VB (Group B) tool. The 150W holmium:YAG cyber Ho generator was used. We compared operative time, dusting time, need for flexible ureteroscopy due to stone push-up and occurrence of ureteral lesions. The stone-free rate (SFR) and the occurrence of postoperative ureteral strictures were assessed. RESULTS: 186 patients were treated, of which 92 with the VT (49.5%, Group A) and 94 with the VB (50.5%, Group B). Mean stone size was 0.92 vs. 0.91 cm in Groups A vs. B (p = 0.32). Mean total operative time and dusting time were comparable between groups. 7 (7.6%) vs. 6 (6.4%) patients in Groups A vs. B required a flexible ureteroscope because of stone push-up (p = 0.12). Ureteral mucosa lesions were observed in 15 (16.3%) vs. 18 (19.1%) cases in the VT vs. VB group (p = 0.09). 1-Month SFR was comparable (97.8% vs. 95.7%, p = 0.41). We observed one case (1.1%) of postoperative ureteral stricture in the VT group vs. two cases (2.1%) in the VB group (p = 0.19). CONCLUSIONS: VT and VB are equally safe and effective tools in reducing retropulsion of ureteral stones. Operative time, dusting time and SFR were comparable. They also equally avoided stone push-up and prevented ureteral lesions, which may later occur in ureteral strictures.

Evaluation of the learning curve for Thulium laser enucleation of the prostate in a contemporary cohort.

PURPOSE: To assess the learning curve of Thulium laser enucleation of the prostate (ThuLEP) of a single surgeon. METHODS: Hundred patients suffering from benign prostatic hyperplasia were treated by the same surgeon. In all cases, a well-trained urologist was present in the operating room. Patients urinary function was assessed preoperatively using the International Prostate Symptoms Score (IPSS), maximum flow rate and Post-Void Residual volume. Preoperative prostate volume was recorded. Enucleation and morcellation efficiency and complication rate were evaluated. Patients were divided into 5 cohorts of 20 consecutive cases to assess changes in outcomes through time. RESULTS: Mean age of patients was 73.1 years (SD 17.5) and mean prostate volume was 89.7 ml (SD 55.1). Overall, mean enucleation and morcellation efficiency were 1.7 (SD 2.9) and 5.1 (SD 2.7) g/min. A statistically significant increase in enucleation efficiency was observed when comparing cohort 1 vs 2 (0.9 vs 1.3 g/min, p = 0.03) and cohort 2 vs 3 (1.3 vs 1.7 g/min, p = 0.02). A statistically significant increase in morcellation efficiency was observed when comparing cohort 1 vs 2 (2.8 vs 3.7 g/min, p = 0.02) and cohort 2 vs 3 (3.7 vs 4.9 g/min, p = 0.03). In both cases, no significant differences were observed when comparing the following cohorts. Complication rate showed no significant differences throughout the caseload. CONCLUSIONS: In our single-surgeon experience, we observed a learning curve of nearly 60 cases for the ThuLEP procedure in presence of a well-trained surgeon. Complication rate was low from the beginning of surgical experience.

The recombinant alpha subunit of glutamate synthase: spectroscopic and catalytic properties.

As part of our studies of Azospirillum brasilense glutamate synthase, a complex iron-sulfur flavoprotein, we have overproduced the two enzyme subunits separately in Escherichia coli. The beta subunit (53.2 kDa) was demonstrated to contain the site of NADPH oxidation of glutamate synthase and the FAD cofactor, which was identified as Flavin 1 of glutamate synthase, the flavin located at the site of NADPH oxidation. We now report the overproduction of the glutamate synthase alpha subunit (162 kDa), which is purified to homogeneity in a stable form. This subunit contains FMN as the flavin cofactor which exhibits the properties of Flavin 2 of glutamate synthase: reactivity with sulfite to yield a flavin-N(5)-sulfite addition product (Kd = 2.6 +/- 0.22 mM), lack of reactivity with NADPH, reduction by L-glutamate, and reoxidation by 2-oxoglutarate and glutamine. Thus, FMN is the flavin located at the site of reduction of the iminoglutarate formed on the addition of glutamine amide group to the C(2) carbon of 2-oxoglutarate. The glutamate synthase alpha subunit contains the [3Fe-4S] cluster of glutamate synthase, as shown by low-temperature EPR spectroscopy experiments. The glutamate synthase alpha subunit catalyzes the synthesis of glutamate from L-glutamine and 2-oxoglutarate, provided that a reducing system (dithionite and methyl viologen) is present. The FMN moiety but not the [3Fe-4S] cluster of the subunit appears to participate in this reaction. Furthermore, the isolated alpha subunit of glutamate synthase exhibits a glutaminase activity, which is absent in the glutamate synthase holoenzyme. These findings support a model for glutamate synthase according to which the enzymes prepared from various sources share a common glutamate synthase function (the alpha subunit of the bacterial enzyme, or its homologous polypeptide forming the ferredoxin-dependent plant enzyme) but differ for the chosen electron donor. The pyridine nucleotide-dependent forms of the enzyme have recruited a FAD-dependent oxidoreductase (the bacterial beta subunit) to mediate electron transfer from the NAD(P)H substrate to the glutamate synthase polypeptide. However, it appears that the presence of the enzyme beta subunit and/or of the additional iron-sulfur clusters (Centers II and III) of the bacterial glutamate synthase is required for communication between Center I (the [3Fe-4S] center) and the FMN moiety within the alpha subunit, and for ensuring coupling of glutamine hydrolysis to the transfer of the released ammonia molecule to 2-oxoglutarate in the holoenzyme.

Properties of the recombinant beta subunit of glutamate synthase.

Glutamate synthase is a complex iron-sulfur flavoprotein containing one molecule each of FAD and FMN and three distinct iron-sulfur centers/alpha beta protomer. Production of the beta subunit was observed in total extracts of Escherichia coli BL21 (DE) cells harbouring a pT7-7 derivative carrying gltD, the gene encoding the Azospirillum brasilense glutamate synthase beta subunit. The protein was soluble, and the identity of the purified protein with the Azospirillum glutamate synthase beta subunit was confirmed by N-terminal sequence analysis. The kinetic and spectroscopic characterization of the glutamate synthase beta subunit confirmed that it contains the NADPH binding site, but, in contrast with earlier proposals that assigned both FAD and FMN binding sites to the alpha subunit of glutamate synthase, the beta subunit was shown to contain stoichiometric amounts of FAD. No iron-sulfur centers were detected by EPR spectroscopy measurements of the recombinant beta subunit. Under steady-state conditions, the glutamate synthase beta subunit can catalyze the NADPH-dependent reduction of several synthetic electron acceptors but no glutamate synthase or glutamate dehydrogenase reactions in either direction. The results are in agreement with previous data from our laboratory and, together with the absence of amino acid sequence similarity between glutamate synthase beta subunit and glutamate dehydrogenases, are against the hypothesis that glutamate synthase is evolutionarily derived from the association of an ancestral glutamate dehydrogenase (the beta subunit) and an amidotransferase (the alpha subunit). The protein-bound FAD is reduced by NADPH at a rate much faster than turnover with synthetic electron acceptors, leading to formation of a stable reduced flavin-NADP+ charge-transfer complex. The rate of reduction of the bound FAD by NADPH is also similar to the rate at which one of the flavins is reduced in the native glutamate synthase, as measured in a stopped-flow spectrophotometer under pre-steady-state conditions. The ability of FAD bound to the beta subunit of glutamate synthase to react with NADPH and the lack of reactivity with sulfite lead us to conclude that FAD is Flavin 1 of glutamate synthase [Vanoni, M.A., Edmondson, D.E., Zanetti, G. & Curti, B. (1992) Biochemistry 31, 4613-4623].

In the budding yeast Kluyveromyces marxianus, adenylate cyclase is regulated by Ras protein(s) in vitro.

The presence of adenylate cyclase activity was first demonstrated in membrane fractions from the budding yeast Kluyveromyces marxianus. The enzyme showed a Mn(2+)- and Mg(2+)-dependent activity, with optimal pH at around 6 as observed in other yeast species. As in Saccharomyces cerevisiae, where adenylate cyclase is regulated by RAS1 and RAS2, we detected a guanyl nucleotide-dependent activity. Interestingly Y13-259 monoclonal antibody, raised against mammalian p21Ha-ras, inhibited Mg2+ plus GTP-gamma-S-dependent cAMP production, suggesting that the GTP binding proteins involved in adenylate cyclase regulation could be Ras proteins. The same antibody recognized on Western blot and immunoprecipitated a 40 kDa polypeptide from K. marxianus crude membranes. This polypeptide was not detected by an anti-RAS2 polyclonal antibody raised against S. cerevisiae RAS2 protein, suggesting that Ras proteins from the two species could be structurally different.