Systemic tobramycin concentrations during selective decontamination of the digestive tract in intensive care unit patients on continuous venovenous hemofiltration

OBJECTIVE: To study whether selective decontamination of the digestive tract (SDD) results in detectable serum tobramycin concentrations in intensive care unit (ICU) patients with acute renal failure treated with continuous venovenous hemofiltration (CVVH). DESIGN AND SETTING: Prospective, observational, single-center study in a mixed medical-surgical ICU. PATIENTS: Adult ICU patients receiving SDD for at least 3 days and being treated with CVVH because of acute renal failure. MEASUREMENTS AND RESULTS: Tobramycin serum concentrations were measured at the 3rd day after start of CVVH and every 3 days thereafter. Detectable serum concentrations of tobramycin were found in 12 (63%) of 19 patients and in 15 (58%) of the 26 samples. With a toxic tobramycin concentration defined as more than 2.0 mg/l, we found one patient with a toxic concentration of 3.0 mg/l. In three other patients tobramycin concentrations of >or=1.0 mg/l were found. CONCLUSIONS: In patients with acute renal failure treated with CVVH, administration of SDD with tobramycin can lead to detectable and potentially toxic serum tobramycin concentration

Patient-derived mutations within the N-terminal domains of p85α impact PTEN or Rab5 binding and regulation

The p85α protein regulates flux through the PI3K/PTEN signaling pathway, and also controls receptor trafficking via regulation of Rab-family GTPases. In this report, we determined the impact of several cancer patient-derived p85α mutations located within the N-terminal domains of p85α previously shown to bind PTEN and Rab5, and regulate their respective functions. One p85α mutation, L30F, significantly reduced the steady state binding to PTEN, yet enhanced the stimulation of PTEN lipid phosphatase activity. Three other p85α mutations (E137K, K288Q, E297K) also altered the regulation of PTEN catalytic activity. In contrast, many p85α mutations reduced the binding to Rab5 (L30F, I69L, I82F, I177N, E217K), and several impacted the GAP activity of p85α towards Rab5 (E137K, I177N, E217K, E297K). We determined the crystal structure of several of these p85α BH domain mutants (E137K, E217K, R262T E297K) for bovine p85α BH and found that the mutations did not alter the overall domain structure. Thus, several p85α mutations found in human cancers may deregulate PTEN and/or Rab5 regulated pathways to contribute to oncogenesis. We also engineered several experimental mutations within the p85α BH domain and identified L191 and V263 as important for both binding and regulation of Rab5 activit

Modeling of Molecular Interaction between Apoptin, BCR-Abl and CrkL - An Alternative Approach to Conventional Rational Drug Design

In this study we have calculated a 3D structure of apoptin and through modeling and docking approaches, we show its interaction with Bcr-Abl oncoprotein and its downstream signaling components, following which we confirm some of the newly-found interactions by biochemical methods. Bcr-Abl oncoprotein is aberrantly expressed in chronic myelogenous leukaemia (CML). It has several distinct functional domains in addition to the Abl kinase domain. The SH3 and SH2 domains cooperatively play important roles in autoinhibiting its kinase activity. Adapter molecules such as Grb2 and CrkL interact with proline-rich region and activate multiple Bcr-Abl downstream signaling pathways that contribute to growth and survival. Therefore, the oncogenic effect of Bcr-Abl could be inhibited by the interaction of small molecules with these domains. Apoptin is a viral protein with well-documented cancer-selective cytotoxicity. Apoptin attributes such as SH2-like sequence similarity with CrkL SH2 domain, unique SH3 domain binding sequence, presence of proline-rich segments, and its nuclear affinity render the molecule capable of interaction with Bcr-Abl. Despite almost two decades of research, the mode of apoptin’s action remains elusive because 3D structure of apoptin is unavailable. We performed in silico threedimensional modeling of apoptin, molecular docking experiments between apoptin model and the known structure of Bcr- Abl, and the 3D structures of SH2 domains of CrkL and Bcr-Abl. We also biochemically validated some of the interactions that were first predicted in silico. This structure-property relationship of apoptin may help in unlocking its cancer-selective toxic properties. Moreover, such models will guide us in developing of a new class of potent apoptin-like molecules with greater selectivity and potency