Rational engineering of human cytochrome P450 2B6 for enhanced expression and stability: importance of a Leu264->Phe substitution

Despite the emerging importance of human P450 2B6 in xenobiotic metabolism, thorough biochemical and biophysical characterization has been impeded as a result of low expression in Escherichia coli. Comparison with similar N-terminal truncated and C-terminal His-tagged constructs (rat P450 2B1dH, rabbit 2B4dH, and dog 2B11dH) revealed that P450 2B6dH showed the lowest thermal stability, catalytic tolerance to temperature, and chemical stability against guanidinium chloride-induced denaturation. Eleven P450 2B6dH mutants were rationally engineered based on sequence comparison with the three other P450 2B enzymes and the solvent accessibility of residues in the ligand-free crystal structure of P450 2B4dH. L198M, L264F, and L390P showed approximately 3-fold higher expression than P450 2B6dH. L264F alone showed enhanced stability against thermal and chemical denaturation compared with P450 2B6dH and was characterized further functionally. L264F showed similar preferential inhibition by pyridine over imidazole derivatives as P450 2B6dH. The Leu(264)-->Phe substitution did not alter the K(s) for inhibitors or the substrate benzphetamine, the K(m) for 7-ethoxy-4-(trifluoromethyl)coumarin, or the benzphetamine metabolite profiles. The enhanced stability and monodisperse nature of L264F made it suitable for isothermal titration calorimetry studies. Interaction of 1-benzylimidazole with L264F yielded a clear binding isotherm with a distinctly different thermodynamic signature from P450 2B4dH. The inhibitor docked differently in the binding pocket of a P450 2B6 homology model than in 2B4, highlighting the different chemistry of the active site of these two enzymes. Thus, L264F is a good candidate to further explore the unique structure-function relationships of P450 2B6 using X-ray crystallography and solution thermodynamics.     

Prognostic significance of Her2/neu overexpression in patients with muscle invasive urinary bladder cancer treated with radical cystectomy

Introduction The aim of the study was to evaluate the status of Her2/neu protein expression in patients with muscle-invasive urothelial carcinomas of the bladder treated with radical cystectomy and to determine its prognostic significance. Material and methods We retrospectively analyzed the data of 90 patients who had undergone cystectomy for invasive transitional cell carcinoma of the urinary bladder. Immunohistochemical analysis for Her2/neu was done on paraffin-fixed tissues with CB11 antibodies (BioGenex, San Ramon, CA, USA). Sections with grade 2 and grade 3 staining were considered positive for Her2/neu. Results Over a median follow-up period of 46 months (24–96 months) 46 patients are living without disease recurrence and six with recurrent disease either at the local site or with distant metastases. The remaining 38 patients have died. The median overall survival time was 50 months, and median disease-free survival time was 40 months. The Her2/neu status was significantly related to the tumor stage (P = 0.001), lymph node involvement (77% in N+ vs 23% in N0; P = 0.001) and the grade of the disease (32% of grade 2 vs 71% of grade 3; P = 0.037). Kaplan–Meier curves showed a significantly worse disease-related survival period (log rank P = 0.011) for patients with Her2 overexpressing tumors than for those without overexpression. In addition to tumor stage [P = 0.001; relative risk (RR) = 2.62] and lymph node status (P = 0.0001; RR = 2.95), Her2 status (P = 0.020; RR = 2.22) was identified as an independent predictor for disease-related survival in a multivariate analysis. Conclusion These results suggest that Her2 expression might provide additional prognostic information for patients with muscle-invasive bladder cancer. Future studies on Her2 expression with chemosensitivity and the efficacy of Her2-targeted therapies in urothelial carcinomas are warranted.     

Evaluation of laparoscopic radical cystectomy for loco-regionally advanced bladder cancer

We studied the safety and feasibility of laparoscopic radical cystectomy (LRC) in patients with loco-regionally advanced bladder cancer and report the short-term oncological outcome. This study comprised a total of 13 patients (10 males, 3 females), who presented with myriad of symptoms and on imaging they were found to have radiologically evident advanced disease (6 pelvic lymphadenopathies, 10 extravesical tumor extensions, three prostate/seminal vesical invasions). In view of recalcitrant symptoms (hematuria, frequency and irritative voiding) all patients underwent LRC and bilateral modified pelvic lymphadenectomy with ileal conduit urinary diversion. Mean age of the patients was 56.3 years. Mean operative time was 310 min with an average blood loss of 556 ml. No major intra-operative complications were noted. One patient died in the post-operative period due to sepsis. Histopathology report revealed pT3b N0 in two patients; pT3b N1 in four; pT3b N2 in three; pT4a N0 in one, and pT4aN1 in three patients. Adjuvant chemotherapy was administered in nine patients. At mean follow up of 18 months (range 6–28), seven patients are alive and cancer-free, while two patients are alive with metastases. LRC provides an alternative approach for treatment of patients with loco-regionally advanced bladder cancer, who suffer from recurrent hematuria and severe irritative voiding symptoms, in whom open surgery was the standard approach hitherto. However, it should be considered experimental and should be attempted only by surgeons who have significant experience of laparoscopic pelvic surgery and advanced skills, and after discussing the potential risks and benefits with the patient.     

The MLC1v gene provides a transgenic marker of myocardium formation within developing chambers of the Xenopus heart.

Many details of cardiac chamber morphogenesis could be revealed if muscle fiber development could be visualized directly within the hearts of living vertebrate embryos. To achieve this end, we have used the active promoter of the MLC1v gene to drive expression of green fluorescent protein (GFP) in the developing tadpole heart. By using a line of Xenopus laevis frogs transgenic for the MLC1v-EGFP reporter, we have observed regionalized patterns of muscle formation within the ventricular chamber and maturation of the atrial chambers, from the onset of chamber formation through to the adult frog. In f1 generation MLC1v-EGFP animals, promoter activity is first detected within the looping heart tube and delineates the forming ventricular chamber and proximal outflow tract throughout their development. The 8-kb MLC1v promoter faithfully reproduces the embryonic expression of the endogenous MLC1v mRNA. At later larval stages, weak patches of EGFP fluorescence are found on the atrial side of the atrioventricular boundary. Subsequently, an extensive lattice of MLC1v-expressing fibers extend across the mature atrial chambers of adult frog hearts and the transgene reveals the differing arrangement of muscle fibers in chamber versus outflow myocardium. The complete activity of the promoter resides within the proximal 4.5 kb of the MLC1v DNA fragment, whereas key elements regulating chamber-specific expression are present in the proximal-most 1.5 kb. Finally, we demonstrate how cardiac and craniofacial muscle expression of the MLC1v promoter can be used to diagnose mutant phenotypes in living embryos, using the injection of RNA encoding a Tbx1-engrailed repressor-fusion protein as an example.     

Formulation and evaluation of in situ gelling system of dimenhydrinate for nasal administration

Nasal drug delivery has a variety of advantages. Drugs can be rapidly absorbed through the nasal mucosa, giving rapid onset of action, and avoiding presystemic metabolism. In present study; the nasal mucoadhesive in situ gels of anti-emetic drug Dimenhydrinate were formulated using Gellan gum and Carbopol 934P. The in situ gels so prepared were characterized for gelation, viscosity, gel strength, mucoadhesion, drug content, drug diffusion, ex vivo permeation and histopathological studies. The optimized formulation passing from above tests was further subjected to accelerated stability study. It retained the good stability over the period of 90 days. From the overall performance this in situ gel seems to be an effective delivery system for the nasal route.     

Lipoma of Columella with septal extension in Pai syndrome: report of a rare case

Background

Lipoma in nasal region in a case of Pai syndrome is very rare. Very few cases of Pai syndrome with columellar lipoma with extension to septum and tip have been reported till date.

Case presentation

We report a very rare form of nasal lipoma in a case of Pai syndrome with extension to septum and tip in a 4 year old female child.

Conclusions

Pai syndrome with columellar lipoma with septal and tip extension should always be kept in mind when evaluating a swelling in the region of nasal tip, collumella and septum with notch in upper lip and corpus callosal lipoma.

     

Lung tattooing combined with immediate video-assisted thoracoscopic resection (IVATR) as a single procedure in a hybrid room: our institutional experience in a pediatric population

Background

Analysis of small pulmonary nodules in children poses an important diagnostic and therapeutic challenge for clinicians.

Objective

To review our experience of lung tattooing with immediate video-assisted thoracoscopic resection (IVATR) performed as a single procedure in a hybrid room for technical difficulties, complications and diagnostic yield of the procedure.

Material and methods

Retrospective analysis of 31 children (16 boys, 15 girls) who underwent lung tattooing of various lesions from January 2001 to July 2011. Data were collected from the Interventional Radiology database, Electronic Patient Chart (EPC) and PACS.

Results

A total of 34 lesions were treated in 31 children. Tattooing was performed on lung lesions with median size 3 mm and median depth 2 mm from pleura. Technical success was 91.1% and diagnostic yield was 100%. In seven children, it was combined with other interventional radiologic procedures. The median procedure time for lung tattooing and IVATR was 197 min.

Conclusion

Lung tattooing with IVATR as a single procedure in a hybrid room is safe and effective in children with several inherent advantages, including avoiding the need to move the child from the interventional radiology suite to the operating room.     

Prospective, Pilot, Open-Label, Short-Term Study of Conversion to Leflunomide Reverses Chronic Renal Allograft Dysfunction

Leflunomide (LEF) is a synthetic isoxazole derivative with anti-inflammatory and antiviral properties, which has been reported to prevent acute rejection and delay progression of chronic allograft nephropathy (CAN) in animal models. We performed a pilot, crossover trial in 22 renal transplant recipients who were converted from azathioprine (AZA) or mycophenolate mofetil (MMF) to LEF in an effort to slow progression of renal dysfunction [deteriorating renal function (n = 5), cyclosporine (CyA) nephrotoxicity (n = 4) or biopsy-proven CAN (n = 13)]. Baseline maintenance immunosuppression consisted of CyA, AZA or MMF and prednisone. Six-month postconversion patient and graft survival was 100% and 91%, respectively. Mean serum creatinine 6months preconversion was 2.2 +/- 0.6mg/dL, at initiation was 3.0 +/- 1.1 mg/dL, and 6 months postconversion was 2.8 +/- 1.3 mg/dL. The rate of change in serum creatinine was 35 +/- 39%/6 months preconversion and -5 +/- 21%/6 months postconversion to LEF (p = 0.003). Two patients discontinued LEF for diarrhea and myalgia. No readmissions, increase in liver function tests, infections or acute rejection episodes occurred. Mean CyA levels did not change, 146 +/- 72 ng/ mL pre-LEF vs. 132 +/- 51 ng/mL post-LEF, p = NS. Conversion to LEF reversed progression of chronic renal allograft dysfunction with minimal toxicity.