Crystal structure of the Geobacillus stearothermophilus carboxylesterase Est55 and its activation of prodrug CPT-11

Several mammalian carboxylesterases were shown to activate the prodrug irinotecan (CPT-11) to produce 7-ethyl-10-hydroxycamptothecin (SN-38), a topoisomerase inhibitor used in cancer therapy. However, the potential use of bacterial carboxylesterases, which have the advantage of high stability, has not been explored. We present the crystal structure of the carboxyesterase Est55 from Geobacillus stearothermophilus and evaluation of its enzyme activity on CPT-11. Crystal structures were determined at pH 6.2 and pH 6.8 and resolution of 2.0 A and 1.58 A, respectively. Est55 folds into three domains, a catalytic domain, an alpha/beta domain and a regulatory domain. The structure is in an inactive form; the side-chain of His409, one of the catalytic triad residues, is directed away from the other catalytic residues Ser194 and Glu310. Moreover, the adjacent Cys408 is triply oxidized and lies in the oxyanion hole, which would block the binding of substrate, suggesting a regulatory role. However, Cys408 is not essential for enzyme activity. Mutation of Cys408 showed that hydrophobic side-chains were favorable, while polar serine was unfavorable for enzyme activity. Est55 was shown to hydrolyze CPT-11 into the active form SN-38. The mutant C408V provided a more stable enzyme for activation of CPT-11. Therefore, engineered thermostable Est55 is a candidate for use with irinotecan in enzyme-prodrug cancer therapy.     

Numerical Modeling of Intraventricular Flow during Diastole after Implantation of BMHV

This work presents a numerical simulation of intraventricular flow after the implantation of a bileaflet mechanical heart valve at the mitral position. The left ventricle was simplified conceptually as a truncated prolate spheroid and its motion was prescribed based on that of a healthy subject. The rigid leaflet rotation was driven by the transmitral flow and hence the leaflet dynamics were solved using fluid-structure interaction approach. The simulation results showed that the bileaflet mechanical heart valve at the mitral position behaved similarly to that at the aortic position. Sudden area expansion near the aortic root initiated a clockwise anterior vortex, and the continuous injection of flow through the orifice resulted in further growth of the anterior vortex during diastole, which dominated the intraventricular flow. This flow feature is beneficial to preserving the flow momentum and redirecting the blood flow towards the aortic valve. To the best of our knowledge, this is the first attempt to numerically model intraventricular flow with the mechanical heart valve incorporated at the mitral position using a fluid-structure interaction approach. This study facilitates future patient-specific studies.     

Hypoxia-induced DNp73 stabilization regulates Vegf-A expression and tumor angiogenesis similar to TAp73

P73, the homolog of p53, exists in 2 major forms: either as a pro-apoptotic TAp73 or an amino-terminally truncated DNp73, the latter lacking the first transactivation domain. While TAp73s tumor suppressive functions have been established, DNp73 is an anti-apoptotic protein conferring chemoresistance and is associated with poor survival. However, both forms are variably overexpressed in many human cancers. In this context, we have recently demonstrated that TAp73 is stabilized by hypoxia, a tumor-relevant condition that is associated with cell survival, via HIF-1α-mediated suppression of Siah1 E3 ligase that degrades TAp73. Consequently, hypoxic signals lead to TAp73-mediated activation of several angiogenic genes and blood vessel formation, thereby supporting tumorigenesis. We show here that, similar to TAp73, DNp73 is stabilized by hypoxia in a HIF-1α-dependent manner, which otherwise is degraded by Siah1. Moreover, DNp73 is capable of inducing the expression of Vegf-A, the prototypic angiogenic gene, and loss of DNp73 expression results in reduction in tumor vasculature and size. These data therefore indicate a common mode of regulation for both p73 forms by hypoxia, resulting in the promotion of angiogenesis and tumor growth, highlighting common functionality of these antagonistic proteins under specific physiological contexts.     

The active ring-like structure of SecA revealed by electron crystallography: conformational change upon interaction with SecB

SecA is a multifunctional protein involved in protein translocation in bacteria. The structure of SecA on membrane is dramatically altered compared with that in solution, accompanying with functional changes. We previously reported the formation of a novel ring-like structure of SecA on lipid layers, which may constitute part of the preprotein translocation channel. In the present work, two-dimensional crystallization of Escherichia coli SecA on lipid monolayers was performed to reveal the structural details of SecA on lipid layers and to investigate its function. The 2D crystals composed of ring-like structures were obtained by specific interaction between SecA and negatively charged lipid. The 2D projection map and 3D reconstruction from negative stained 2D crystals exhibited a distinct open channel-like structure of SecA, with an outer diameter of 7 nm and an inner diameter of 2 nm, providing the structural evidence for SecA importance in forming the part of the translocation channel. This pore structure is altered after transferring crystals to the SecB solution, indicating that the lipid-specific SecA structure has the SecB binding activity. The strategy developed here provides a promising technique for studying structure of SecA complex with its ligand on membrane.     

Intergeneric and Interspecific Relationships in Araceae tribe Caladieae and Development of Molecular Markers using Amplified Fragment Length Polymorphism (AFLP)

Since amplified fragment length polymorphism (AFLP) analysishas proved useful in distinguishing cultivars of Caladium, itwas used to assess the status of species of Caladium vs. Xanthosoma,both in tribe the Caladieae, and to reassess the position ofHapaline in the same tribe. AFLP analysis using three primercombinations was carried out on four species of Caladium(C.bicolor, C. humboldtii, C. lindenii and C. schomburgkii). Resultsshowed that AFLP can distinguish between the different speciesby their unique and different banding patterns. AFLP analysisconfirmed that C. humboldtii is a species distinct from C. bicolorand that C. lindenii is a true Caladium species and does notbelong to Xanthosoma. UPGMA cluster analysis showed that C.bicolor and C. schomburgkii are most similar and that C. humboldtiiis closer to the C. bicolor / C. schomburgkii cluster comparedwith C. lindenii. Genetic relationships between Caladium, Xanthosoma,Hapaline, Alocasia and Protarum were also examined by AFLP analysisusing eight primer combinations. Several useful molecular markerswere specific either to Caladium orXanthosoma , so that AFLPcan be used to distinguish species of these two genera. Geneticanalysis of the genera examined confirms that the Caladieaeand Colocasieae tribes are distinct and that Hapaline fallswithin the tribe Caladieae and that Protarum is most distantfrom all the genera examined. Copyright 2000 Annals of BotanyCompany Araceae, Caladium, Xanthosoma, Hapaline, Alocasia, Protarum, AFLP DNA fingerprinting, diversity, AFLP markers     

Utilization of oil palm decanter cake for cellulase and polyoses production

The abundance of oil palm decanter cake (OPDC) is a problem in oil palm mills. However, this lignocellulosic biomass can be utilized for cellulase and polyoses production. The effectiveness of chemical and physical pretreatment in reducing the lignin content was studied by saccharification using a Celluclast 1.5 L and scanning electron microscope. Physicochemical pretreatment of OPDC with 1% (w/v) NaOH and autoclaving at 121°C for 20 min increased potential polyoses produced to 52.5% and removed 28.7% of the lignin content. The optimized conditions for cellulase production by a locally isolated fungus were a time of 120 h, a substrate of untreated OPDC, a spore concentration of 1 × 10⁷ spore/mL, a temperature of 30°C, and a pH between 7.0 and 7.5. Trichoderma asperellum UPM1 produced carboxymethylcellulase (CMCase), ??-glucosidase and filter paper activity (FPase) in the following concentrations: 17.35, 0.53, and 0.28 U/mL, respectively. Aspergillus fumigatus UPM2 produced the CMCase, ??-glucosidase and FPase in the following amounts: 10.93, 0.76, and 0.24 U/mL. The cellulases from T. asperellum UPM1 produced 2.33 g/L of polyoses and the cellulases from A. fumigatus UPM2 produced 4.37 g/L of polyoses.     

Use of an algal consortium of five algae in the treatment of landfill leachate using the high-rate algal pond system

Five species of microalgae Chlorella vulgaris, Scenedesmus quadricauda, Euglena gracilis, Ankistrodesmus convolutus and Chlorococcum oviforme, were screened for their ability to grow in treated landfill leachate (TL) using shake flask cultures. The treated leachate had undergone previous treatment through mechanical aeration in treatment ponds at the landfill site. The five algae, except for C. oviforme, were able to grow in medium containing up to 50% TL. Two high-rate algal ponds (HRAP) equipped with paddlewheel were used for the semi-continuous cultures. A mixture of the five algae was used to inoculate one of two HRAPs for secondary treatment of TL. The other HRAP was filled with natural lake water containing mixed populations of algae. A volume of 400?mL (1%) from both ponds were removed daily and replaced with TL. The leachate loading rate was increased to 2% (0.8?L?day^?1) on day?197 and then to 4% (1.6?L?day^?1) on day?309, providing hydraulic retention time of 100, 50 and 25?days, respectively. Although higher biomass was obtained in the HRAP containing the consortium of five algae, there was no significant difference in reduction of pollutants between the two ponds. The HRAPs produced algal biomass ranging from 2.00 to 5.54?g dry weight?L^?1 with significant reduction in chemical oxygen demand (91.0%), ammoniacal nitrogen (99.9%) and orthophosphate (86.0%) contents. The HRAP offers a potential treatment system for TL which is simple, low cost, flexible in use and requiring low maintenance.     

Long-Term Vector Integration Site Analysis Following Retroviral Mediated Gene Transfer to Hematopoietic Stem Cells for the Treatment of HIV Infection

We previously reported the efficacy of nonmyeloablative allogeneic transplantation in 2 HIV positive recipients, one of whom received retrovirus transduced hematopoietic stem cells to confer resistance to HIV. Here we report an assessment of retroviral integration sites (RISs) recovered out to 3 years post-transplantation. We identified 213 unique RISs from the patient''s peripheral blood samples by linear amplification-mediated PCR (LAM-PCR). While vector integration patterns were similar to that previously reported, only 3.76% of RISs were common among early (up to 3 months) and late samples (beyond 1 year). Additionally, common integration sites were enriched among late samples (14.9% vs. 36.8%, respectively). Three RISs were found near or within known oncogenes, but 2 were limited to early timepoints. Interestingly, an integration site near the MDS1 gene was detected in long-term follow-up samples; however, the overall contribution of MDS1 integrated clone remained stably low during follow-up.